Nucleic acids encoding Fas associated proteins and screening assays using same

ABSTRACT

The present invention provides mammalian protein tyrosine phosphatases, human PTP-BAS type 4, human PTP-BAS type 5a and mouse PTP-BAS type 5b, each of which is a Fas-associated protein (FAP), nucleic acid molecules encoding a PTP-BAS type 4 or a PTP-BAS type 5 and antibodies specific for a PTP-BAS type 4 or for a PTP-BAS type 5. The invention also provides methods for identifying FAP&#39;s, which can associate with Fas and can modulate apoptosis. The invention also provides screening assays for identifying an agent that can effectively alter the association of a FAP with Fas and, therefore, can increase or decrease the level of apoptosis in a cell. The invention further provides methods of modulating apoptosis in a cell by introducing into the cell a nucleic acid molecule encoding a PTP-BAS or an antisense nucleotide sequence, which is complementary to a portion of a nucleic acid molecule encoding a PTP-BAS. The invention also provides a method of using a reagent that can specifically bind to a FAP to diagnose a pathology that is characterized by an increased or decreased level of apoptosis in a cell. The invention also provides methods of modulating apoptosis in a cell by contacting the cell with an agent that effectively alters the association of a FAP and Fas in a cell or alters the activity of a FAP in a cell.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of molecular biology andmolecular medicine and more specifically to the identification ofproteins involved in programmed cell death and associations of theseproteins.

2. Background Information

Programmed cell death is a physiologic process that ensures homeostasisis maintained between cell production and cell turnover in essentiallyall self-renewing tissues. In many cases, characteristic morphologicalchanges, termed "apoptosis," occur in a dying cell. Since similarchanges occur in different types of dying cells, cell death appears toproceed through a common pathway in different cell types.

In addition to maintaining tissue homeostasis, apoptosis also occurs inresponse to a variety of external stimuli, including growth factordeprivation, alterations in calcium levels, free-radicals, cytotoxiclymphokines, infection by some viruses, radiation and mostchemotherapeutic agents. Thus, apoptosis is an inducible event thatlikely is subject to similar mechanisms of regulation as occur, forexample, in a metabolic pathway. In this regard, dysregulation ofapoptosis also can occur and is observed, for example, in some types ofcancer cells, which survive for a longer time than corresponding normalcells, and in neurodegenerative diseases where neurons die prematurely.In viral infections, induction of apoptosis can figure prominently inthe pathophysiology of the disease process.

Some of the proteins involved in programmed cell death have beenidentified and associations among some of these proteins have beendescribed. However, the mechanisms by which these proteins mediate theiractivity remains unknown. The identification of the proteins involved incell death and an understanding of the associations between theseproteins can provide a means for manipulating the process of apoptosisin a cell and, therefore, selectively regulating the relative lifespanof a cell.

A cell surface protein known as Fas (also called APO-1 and CD95;hereinafter "Fas"), which is expressed on various types of human cells,including breast, colon, prostate and pancreatic cancer cells, cantrigger apoptosis. Fas is a member of the tumor necrosis factor receptor(TNFR) family of proteins, which also includes, for example, the nervegrowth factor receptor. These receptor proteins transduce extracellularsignals into a cell and, as a result, can induce cell death or promotecell survival. However, the mechanism by which cell surface receptorssuch as Fas regulate cell death is not known.

Since Fas is present on the cell surface, its action likely is mediatedby Fas binding to one or more intracellular proteins, which ultimatelyeffect cell death. The identification of such intracellular proteinsthat can associate with Fas and, therefore, can be involved in apoptosiswould allow for the manipulation of this association as a means tomodulate apoptosis in a cell. Thus, a need exists to identify proteinsthat associate with Fas. The present invention satisfies this need andprovides additional advantages as well.

SUMMARY OF THE INVENTION

The present invention provides Fas-associated proteins (FAP's),designated PTP-BAS type 4 and PTP-BAS type 5, which are alternativelyspliced forms of a protein tyrosine phosphatase that was originallyisolated from basophils (PTP-BAS). In addition, a subfamily of PTP-BAStype 5 proteins is disclosed, including a human PTP-BAS type 5a and amouse PTP-BAS type 5b, which lack a catalytic phosphatase domain. Theinvention also provides nucleic acid molecules encoding a PTP-BAS type 4or a PTP-BAS type 5, vectors containing these nucleic acid molecules andhost cells containing the vectors. The invention also providesantibodies that can specifically bind to a PTP-BAS type 4 or to aPTP-BAS type 5.

The present invention also provides a screening assay useful foridentifying agents that can effectively alter the association of a FAPsuch as a PTP-BAS with Fas. By altering the association of Fas and aFAP, an effective agent can increase or decrease the level of apoptosisin a cell.

The invention also provides methods of altering the activity of a FAPand, consequently, of Fas in a cell, wherein such increased or decreasedactivity of the FAP or of Fas can modulate the level of apoptosis in thecell. For example, the activity of a FAP in a cell can be increased byintroducing into the cell and expressing a nucleic acid sequenceencoding the FAP. In addition, the activity of a FAP in a cell can bedecreased by introducing into the cell and expressing an antisensenucleotide sequence, which is complementary to a portion of a nucleicacid molecule encoding the FAP.

The invention also provides methods for using a reagent that canspecifically bind a FAP or a nucleotide sequence that can bind to anucleic acid molecule encoding a FAP to diagnose a pathology that ischaracterized by an altered level of apoptosis due to an increased ordecreased level of a FAP in a cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a map of plasmid pEG202, which was used to produce LexAfusion proteins for use in the two hybrid assay system. pEG202 containsa gene that confers ampicillin resistance in bacteria (AmpR) and a colE1origin of replication, which allows the plasmid to replicate inbacteria. The plasmid also contains a gene that allows a yeast cellcontaining the plasmid to grow in the absence of histidine (HIS3) andthe yeast 2 micron origin of replication, which allows replication inyeast.

LexA fusion proteins were produced by inserting a cDNA encoding an openreading frame of the cytoplasmic domain of Fas (amino acids 191 to 335)into the EcoRI/BamHI site in the polylinker, which is downstream of thenucleic acid sequence encoding LexA (LexA 202). Cloning was performed soas to maintain the open reading frame of LexA into the inserted sequencesuch that a LexA fusion protein is produced. The nucleic acid encodingthe fusion protein was expressed from an alcohol dehydrogenase promotor(ADH-P) and terminates at the ADH termination site (ADH-term).

FIG. 2 provides a map of plasmid pJG4-5, which was used to produce B42fusion proteins. The plasmid contains a 2 micron (2 μm) yeast origin ofreplication and a gene that allows a yeast cell containing the plasmidto grow in medium lacking tryptophan (TRP1). B42 fusion proteins wereproduced by inserting as shown HeLa cell-derived cDNA sequences, whichcan encode FAP's, into the EcoRI/XhoI sites located downstream of acassette encoding an initiator methionine (ATG), an SV40 nuclearlocalization signal (Nuc. Loc.), the B42 trans-activator domain (B42)and a hemagglutinin HA1 epitope tag. The nucleic acid encoding the B42fusion protein is expressed from a galactose-inducible promotor(GAL1-P). The plasmid also contain an ADH termination signal (ADH-term),which terminates transcription of the sequence encoding the fusionprotein.

FIG. 3 provides a map of plasmid pSH18-34, which contains the reporterlacZ gene encoding β-galactosidase (β-gal). The plasmid contains abacterial origin of replication (ori) and an ampicillin resistance gene(amp). The plasmid also contains a yeast 2 micron (2 μm) origin ofreplication and a gene that allows a yeast cell containing the plasmidto grow in the absence of uracil (URA3). The lacZ gene is linked to agalactose-inducible promotor (GAL1). In addition to galactose,expression of the lacZ gene depends on LexA binding to the LexA operatorsequences (LexA Op's) and trans-activation. pSH18-34 contains 8 LexAoperators (LexA binding sites).

FIG. 4 provides a map of plasmid pBMT-116, which also was used toproduce LexA/Fas fusion proteins. The plasmid contains a bacterialorigin of replication (ori) and an ampicillin resistance gene (amp). Theplasmid also contains a yeast 2 micron (2 μm) origin of replication anda gene that allows a yeast cell containing the plasmid to grow in theabsence of tryptophan (TRP1). A LexA/Fas fusion protein was produced byinserting a nucleotide sequence encoding the cytoplasmic domain of Fasinto the EcoRI/BamHI sites located in the multiple cloning site. Cloningwas performed so as to maintain the open reading frame of LexA into theinserted sequence such that a LexA/Fas fusion protein was produced.

FIG. 5 provides a map of plasmid pVP-16, which was used to producefusion proteins with the VP-16 trans-activation domain. The plasmidcontains a bacterial origin of replication (ori) and an ampicillinresistance gene (amp). f1 is a regulatory element that directsproduction of the antisense strand. The plasmid also contains a yeast 2micron (2 μm) origin of replication and a gene that allows a yeast cellcontaining the plasmid to grow in the absence of leucine (LEU2). VP-16fusion proteins were produced by inserting a cDNA library prepared frommouse embryo MRNA into the NotI restriction endonuclease site, which isdownstream of the ATG start codon and VP-16 coding sequence(Nuc.Loc.VP16). Transcription is initiated from an ADH promotor (ADHp)and is terminated due to an ADH termination sequence (ADHt).

FIG. 6 schematically shows the Fas protein (FAS/APO-1) and variousfusion proteins containing the glutathione-S-transferase protein (GST)linked to a fragment of Fas as follows: A) Fas(191-335); B)Fas(191-290); C) Fas(246-335); D) Fas(246-290); E) Fas(191-320) and F)Fas(321-335). TM indicates the transmembrane region of Fas. Numbersindicated the amino acid position relative to full length Fas protein.

FIG. 7 schematically shows the PTP-BAS protein and indicates theconserved glycine-leucine-glycine-phenylalanine (GLGF SEQ ID NO:20)regions and the catalytic domain in the cytoplasmic domain of a PTP-BAS.Also shown are the homologous regions encoded by two cDNA clonesobtained using the two hybrid assay (pVP16-31 and pVP16-43; see ExampleI), by two cDNA clones obtained from a human brain cDNA library (HFAP10and HFAP20) and by a cDNA clone obtained from a mouse liver cDNA library(MFAP23; mouse PTP-BAS type 5b). Numbers indicate amino acid positionsrelative to human PTP-BAS type 1. C-termini are indicated by "COOH."VLFDK (SEQ ID NO:21) indicates insertions in GLGF2 of HFAP10 (PTP-BAStype 4) and HFAP20 (PTP-BAS type 5a ). The solid box at the C-terminusof HFAP20 and the stipled box at the C-terminus of MFAP23 indicate aminoacid sequences that diverge from the other members of the PTP-BAS familyof proteins. HFAP20 and MFAP23 terminate as shown and, therefore, do notcontain a catalytic phosphatase domain.

FIG. 8 demonstrates the ability of the polypeptide encoded by pVP16-31to associate with various fragments of Fas. A schematic representationof the full length Fas protein is shown. Solid lines below the schematicof Fas indicate various fragments of Fas that were examined for bindingto the polypeptide encoded by pVP16-31. The column labelled"β-galactosidase activity" indicates those fragments that bound (+) thepVP16-31 polypeptide and, as a result, produced a transcriptionallyactive complex in the two hybrid assay (see Example I) or did not bind(-). The column labelled "In vitro binding assay" indicates thosefragments of Fas that bound (+) or did not bind (-) to a GST/HFAP10polypeptide fusion protein.

FIG. 9 provides a map of plasmid pGEX-2TX, which was used to produceGST/Fas fusion proteins. The plasmid contains a bacterial origin ofreplication (ori) and an ampicillin resistance gene (AmpR).Transcription of the sequence encoding the GST fusion protein isregulated from a tac promotor (ptac). LacIq indicates lac repressor. Thevector also encodes a polypeptide sequence encoding thrombin andenterokinase cleavage sites, the FLAG™ inmunstag peptide, a heart musclekinase recognition site (HMK recognition) and various unique restrictionendonuclease sites as indicated.

FIG. 10 is an autoradiograph of a far-western ligand blotting blot ofmouse S49 T cell proteins probed with either a GST/Fas(119-335)(GST/FAS) fusion protein or GST, alone. Molecular weight markers areindicated at the right. Arrow indicate the migration of FAP's, whichspecifically bound GST/Fas(119-335).

FIG. 11 schematically shows the amino acid sequences of six members ofthe PTP-BAS family of proteins, including the newly disclosed humanPTP-BAS type 4 and two members of the subfamily of PTP-BAS type 5proteins, human PTP-BAS type 5a and mouse PTP-BAS type 5b. Deletedsegments in the alternatively spliced forms of PTP-BAS type 2 and type 3are indicated (see Maekawa et al., FEBS Lett. 337:200-206 (1994), whichis incorporated herein by reference). Numbers indicate the amino acidpositions relative to human PTP-BAS type 1. The amino acid sequences ofthe various PTP-BAS isoforms are indicated by the solid line andcorrespond to the domains indicated at the top of the figure (GLGF (SEQID NO:20) is as in FIG. 7). Dashed lines indicate predicted aminosequences for human PTP-BAS type 4, human PTP-BAS type 5a and mousePTP-BAS type 5b. VLFDK (SEQ ID NO:21) indicates the single letter aminoacid code for the insertion in GLGF2 in human PTP-BAS type 4 and humanPTP-BAS type 5a. The solid boxed region of human PTP-BAS type 5a and thestipled box in mouse PTP-BAS type 5b indicate the amino acid sequencesthat diverge from the other members of the PTP-BAS family.

FIG. 12 lists the deduced amino acid sequence (SEQ ID NO:1)corresponding the cloned PTP-BAS type 4 (HFAP10). The single letteramino acid code is used. Numbers indicate the amino acid positionrelative to amino acid 1 in PTP-BAS type 1. Underlined amino acidsindicate GLGF (SEQ ID NO:20) repeats. Bold print and italics indicatethe VLFDK (SEQ ID NO:21) insertion in GLGF2.

FIG. 13 lists the nucleotide sequence (SEQ ID NO:2) of the clonedPTP-BAS type 4 (HFAP10). Numbers indicate the nucleotide positionrelative to the transcription start site of the nucleic acid moleculeencoding PTP-BAS type 1 and the corresponding amino acid position.Underlined nucleotides indicate sequences encoding GLGF repeats. Boldprint and italics indicate the sequences encoding the VLFDK insertion inGLGF2.

FIG. 14 lists the deduced amino acid sequence (SEQ ID NO:3)corresponding the cloned human PTP-BAS type 5a (HFAP20). The singleletter amino acid code is used. Numbers indicate the amino acid positionrelative to amino acid 1 in PTP-BAS type 1. Underlined amino acidsindicate a GLGF (SEQ ID NO:20) repeat. Bold and italics, together,indicate the VLFDK (SEQ ID NO:21) insertion in GLGF2. Italics, alone,indicate the amino acid sequence that diverges from other members of thePTP-BAS family. The * indicates the location of a STOP codon.

FIG. 15 lists the nucleotide sequence (SEQ ID NO:4 and SEQ ID NO:22) ofthe cloned human PTP-BAS type 5a (HFAP20). Numbers indicate thenucleotide position relative to the transcription start site of thenucleic acid molecule encoding PTP-BAS type 1 and the correspondingamino acid position. Underlined nucleotides indicate sequences encodingGLGF (SEQ ID NO:20) repeats. Bold and italic, together, indicate thesequences encoding the VLFDK (SEQ ID NO:21)insertion in GLGF2. Italics,alone, indicate the sequence that diverges from other members of thePTP-BAS family. - - - ! indicates approximately 600 nucleotides thathave not yet been sequenced. Underlining and italics, together, indicateSTOP codon (TGA and TAA) and a polyadenylation signal (AATAAA).

FIG. 16 lists the deduced amino acid sequence (SEQ ID NO:5)corresponding the cloned mouse PTP-BAS type 5b (MFAP23). The singleletter amino acid code is used. Numbers indicate the amino acid positionrelative to amino acid 1 in human PTP-BAS type 1. Italics indicate theamino acid sequence that diverges from other members of the PTP-BASfamily. The * indicates the location of a STOP codon.

FIG. 17 lists the nucleotide sequence (SEQ ID NO:6) of the cloned mousePTP-BAS type 5b (MFAP23). Numbers indicate the nucleotide positionrelative to the transcription start site of the nucleic acid moleculeencoding human PTP-BAS type 1. Italics indicate the sequence thatdiverges from other members of the PTP-BAS family (beginning at position6022). Underlining indicates STOP codons (TAA and TAG).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel Fas-associated proteins (FAP's),designated PTP-BAS type 4 and PTP-BAS type 5, which are alternativelyspliced forms of a protein tyrosine phosphatase that was originallyisolated from basophils (PTP-BAS). Unlike previously described isoformsof PTP-BAS, PTP-BAS types 4 and 5 each contain a 5 amino acid insertionin a sequence that is otherwise conserved among the members of thePTP-BAS family of proteins. In addition, the invention provides asubfamily of PTP-BAS type 5 proteins such as human PTP-BAS type 5a andmouse PTP-BAS type 5b, which diverge from the other members of thePTP-BAS family of proteins at their C-termini. As a result of thissequence divergence, PTP-BAS type 5 proteins lack a catalyticphosphatase domain and, therefore, are non-catalytic forms of PTP-BAS(see FIGS. 7 and 11).

As used herein, the term "Fas-associated protein" or "FAP" means aprotein that can specifically bind to Fas, which is a cell surfacereceptor involved in apoptosis. A FAP can be identified, for example,using the binding assays described in Examples I and II. Various FAP'sare disclosed herein, including the members of the PTP-BAS family ofproteins (FIG. 11) and the 78 kilodalton (kDa) glucose regulated protein(GRP78; described below). Additional FAP's can be identified using themethods disclosed herein.

Although the term "FAP" is used generally, it should be recognized thata FAP that is identified using an assay described herein can be aportion of a protein, which is considered to be a candidate FAP. As usedherein, the term "candidate FAP" refers to a protein that corresponds toa polypeptide sequence that can bind Fas but that consists of only aportion of the full length protein. For example, a FAP such as a PTP-BAStype 4 or a PTP-BAS type 5 can be identified by obtaining cDNA sequencesfrom a cDNA library, expressing the polypeptides encoded by the cDNAsequences and detecting polypeptides that can bind Fas. Although suchpolypeptides are considered FAP's, it is well known that a cDNA sequenceobtained from a cDNA library may not encode the full length protein.Thus, a cDNA can encode a polypeptide such as a PTP-BAS type 4 or aPTP-BAS type 5 that is only a portion of a full length protein but,nevertheless, assumes an appropriate conformation so as to bind Fas.However, in the full length protein, the polypeptide can assume aconformation that does not bind Fas due, for example, to steric blockingof the Fas binding site. Such a full length protein is an example of acandidate FAP. For convenience of discussion, the term "FAP" as usedherein is intended to include a candidate FAP. Thus, a FAP can be aprotein or a polypeptide portion of a protein that can bind Fas.

Since Fas is involved in apoptosis, the association of a FAP with Fascan affect the level of apoptosis in a cell. Fas is a cell surfaceprotein that can trigger apoptosis in a cell (Itoh et al., Cell66:233-243 (1991), which is incorporated herein by reference). Fas is a36 kDa polypeptide that contains 319 amino acids. The cysteine-richextracellular domain of Fas is similar to the p55 and p75 forms of thetumor necrosis factor receptor (TNFR) as well as to other members of theTNFR family, including the nerve growth factor receptor and receptorsdesignated OX40, CD30 and CD40 (Oehm et al., J. Biol. Chem.267:10709-10715 (1992)). These members of the TNFR family of receptorscan transduce extracellular signals that impact cell survival. Thecytoplasmic domain of Fas (amino acids 191 to 335) is highly homologouswith a TNFR and is essential for Fas function. Thus, proteins that canbind to the cytoplasmic domain of Fas can be signal transducingmolecules in a Fas-mediated cell death pathway. The present inventionprovides FAP's, which can associate with the cytoplasmic domain of Fasand, therefore, can be involved in regulating apoptosis.

Fas is expressed on various types of human cells, including solid tumorsof the breast, colon, prostate and pancreas. The ligand for the Fasreceptor has been cloned and is present on cytotoxic T lymphocytes (Sudaet al., Cell 75:1169-1178 (1993)). The presence of the Fas ligand on Tcells indicates that Fas can act as a target that allows effector cellsof the immune system to recognize and attack a cancer cell. Theidentification herein of various FAP's has provided the necessaryinsight into signal transduction pathways controlled by Fas and hasallowed for the development of assays that are useful for identifyingagents that effectively alter the association of a FAP and Fas. Suchagents can be useful, for example, for providing effective therapy for acancer in a subject or for treating an autoimmune disease.

A FAP such as a PTP-BAS type 4 or a PTP-BAS type 5 can be identified bydetecting the association of the FAP with Fas. Such an association canbe identified using an in vivo assay such as a yeast two hybrid assay(see Example I) or an in vitro assay (see Example II). As used herein,the term "associate" or "association" means that a FAP and Fas can bindto each other relatively specifically and, therefore, can form a boundcomplex. In particular, the association of a FAP and Fas is sufficientlyspecific such that the bound complex can form in vivo in a cell or invitro under suitable conditions (see Example II). Other methods fordetermining whether a protein can bind Fas and, therefore, is a FAP areknown and include, for example, equilibrium dialysis.

In a normal cell, a steady state level of association of a FAP and Faslikely occurs. This steady state level of association of a FAP and Fasin a particular cell type can determine the normal level of apoptosis inthat cell type. An increase or decrease in the steady state level ofassociation of a FAP and Fas in a cell can result in an increased ordecreased level of apoptosis in the cell, which can result in apathology in a subject. The normal association of a FAP and Fas in acell can be altered due, for example, to the expression in the cell of avariant FAP or a variant Fas, either of which can compete for bindingwith the FAP that normally binds to Fas in the cell and, therefore, candecrease the association of a FAP and Fas in a cell. The term "variant"is used generally herein to mean a FAP or a Fas that is different fromthe FAP or Fas, respectively, that normally is found in a particularcell type. In addition, the normal association of a FAP and Fas in acell can be increased or decreased due, for example, to contact of thecell with an agent such as a drug that can effectively alter theassociation of a FAP and Fas in a cell.

Several FAP's, including the newly described PTP-BAS types 4 and 5 (seebelow), were identified using the yeast two hybrid system (Fields andSong, Nature 340:245-246 (1989); Vojtek et al., Cell 74:205-214 (1993),each of which is incorporated herein by reference). An in vivotranscription activation assay such as the yeast two hybrid system isparticularly useful for identifying and manipulating the association ofproteins. In addition, the results observed in such an assay likelymirror the events that naturally occur in a cell. Thus, the resultsobtained in such an in vivo assay can be predictive of results that canoccur in a cell in a subject such as a human subject.

A transcription activation assay such as the yeast two hybrid system isbased on the modular nature of transcription factors, which consist offunctionally separable DNA-binding and trans-activation domains. Whenexpressed as separate proteins, these two domains fail to mediate genetranscription. However, transcription activation activity can berestored if the DNA-binding domain and the trans-activation domain arebridged together due, for example, to the association of two proteins.The DNA-binding domain and trans-activation domain can be bridged, forexample, by expressing the DNA-binding domain and trans-activationdomain as fusion proteins (hybrids), provided that the proteins that arefused to the domains can associate with each other. The non-covalentbridging of the two hybrids brings the DNA-binding and trans-activationdomains together and creates a transcriptionally competent complex. Theassociation of the proteins is determined by observing transcriptionalactivation of a reporter gene (see Example I).

The yeast two hybrid systems exemplified herein use various strains ofS. cerevisiae as host cells for vectors that express the hybridproteins. A transcription activation assay also can be performed using,for example, mammalian cells. However, the yeast two hybrid system isparticularly useful due to the ease of working with yeast and the speedwith which the assay can be performed. For example, yeast host cellscontaining a lacZ reporter gene linked to a LexA operator sequence wereused to demonstrate that a PTP-BAS can interact with Fas (Example I).The DNA-binding domain consisted of the LexA DNA-binding domain, whichbinds the LexA promoter, fused to a portion of Fas and thetrans-activation domain consisted of either the B42 acidic region or theVP16 trans-activation domain fused to cDNA sequences, some of whichencoded FAP's. When the LexA domain was non-covalently bridged to atrans-activation domain fused to a FAP, the association of Fas and theFAP activated transcription of the reporter gene.

A FAP also can be identified using an in vitro assay such as an assayutilizing, for example, a glutathione-S-transferase (GST) fusion proteinas described in Example II. Such an in vitro assay provides a simple,rapid and inexpensive method for identifying and isolating a FAP. Suchan in vitro assay is particularly useful in confirming results obtainedin vivo and can be used to characterize specific binding domains of aFAP (see Example II). For example, a GST/Fas fusion protein can beexpressed and can be purified by binding to an affinity matrixcontaining immobilized glutathione. If desired, a sample that cancontain a FAP or active fragments of a FAP can be passed over anaffinity column containing bound GST/Fas and a FAP that binds to Fas canbe obtained. In addition, GST/Fas can be used to screen a cDNAexpression library, wherein binding of the Fas fusion protein to a cloneindicates that the clone contains a cDNA encoding a FAP.

Using these in vitro and in vivo assays, various FAP's have beenidentified, including the newly described human PTP-BAS type 4, humanPTP-BAS type 5a and mouse PTP-BAS type 5b. PTP-BAS is a protein tyrosinephosphatase that originally was cloned from human basophils (Maekawa etal. FEBS Lett. 337:200-206, 1994, which is incorporated herein byreference). Three isoforms of PTP-BAS, which are formed due tonaturally-occurring in-frame deletions that result because ofalternative splicing, were known prior to the present disclosure.PTP-BAS originally was identified by the reversetranscriptase-polymerase chain reaction cloning method using primersthat were directed to sequences that are conserved among proteintyrosine phosphatases. The function of PTP-BAS was not known prior tothe present disclosure, which demonstrates that PTP-BAS can associatewith the cytoplasmic domain of Fas and, in particular, with a C-terminalregion of Fas. The previously described isoforms of PTP-BAS aredesignated type 1 (2,485 amino acids), type 2 (2,466 amino acids) andtype 3 (2,294 amino acids) and, as disclosed herein, are FAP's. PTP-BAStypes 2 and 3 are alternatively spliced forms of PTP-BAS type 1 andcontain deletions of 19 and 191 amino acids, respectively (see FIG. 11).These deletions are located immediately upstream of threeglycine-leucine-glycine-phenylalanine (GLGF) repeats, which arehomologous to a sequence found in guanylate kinases (FIG. 11).

PTP-BAS types 1, 2 and 3 each contain a single protein tyrosinephosphatase catalytic domain at their carboxy termini (see FIG. 11).These three PTP-BAS isoforms each also have an amino terminalmembrane-binding domain that is similar in sequence to a domain presentin several cytoskeleton-associated proteins. In addition, as disclosedherein, PTP-BAS types 1, 2 and 3 can bind Fas (see Example I).

The present invention provides substantially purified mammalian PTP-BAStype 4 and PTP-BAS type 5 proteins, which are alternatively splicedforms of PTP-BAS. Since the PTP-BAS type 4 and type 5 proteins can bindFas, they are FAP's. In contrast to the previously known human PTP-BAStypes 1, 2 and 3, human PTP-BAS type 4 and type 5a contain a 5 aminoacid insertion in the otherwise conserved GLGF2 repeat (see FIG. 11). Inaddition, the C-termini of the members of the subfamily of PTP-BAS type5 proteins diverge in sequence from the other members of the PTP-BASfamily of proteins and, as a result, PTP-BAS type 5 proteins such ashuman PTP-BAS type 5a and mouse PTP-BAS type 5b do not contain acatalytic phosphatase domain (see FIGS. 7 and 11).

The invention provides substantially purified mammalian PTP-BAS type 4comprising substantially the amino acid sequence of human PTP-BAS type 4shown in FIG. 12 (SEQ ID NO:1), which was derived from the nucleotidesequence shown in FIG. 13 (SEQ ID NO:2). In addition, the inventionprovides a subfamily of substantially purified mammalian PTP-BAS type 5proteins comprising, for example, substantially the amino acid sequenceof human PTP-BAS type 5a shown in FIG. 14 (SEQ ID NO:3), which wasderived from the nucleotide sequence shown in FIG. 15 (SEQ ID NO:4andSEQ ID NO:22), or substantially the amino acid sequence of mouse PTP-BAStype 5b shown in FIG. 16 (SEQ ID NO:5), which was derived from thenucleotide sequence shown in FIG. 17 (SEQ ID NO:6). PTP-BAS type 4 andtype 5 proteins have been characterized as members of the PTP-BAS familyof proteins based on their homology with other members of this proteinfamily (see FIGS. 7 and 11; see, also, Examples III and IV).

As used herein, the term "substantially the amino acid sequence" meansthe disclosed amino acid sequence for human PTP-BAS type 4 (SEQ IDNO:1), a human PTP-BAS type 5a (SEQ ID NO:3) or a mouse PTP-BAS type 5b(SEQ ID NO: 5), as well as amino acid sequences that are similar to SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5, respectively, but have one or moreamino acid additions, deletions or substitutions that do notsubstantially alter the ability of the encoded protein to function likea PTP-BAS type 4 or a PTP-BAS type 5 and, for example, bind Fas. As usedherein, the term "substantially purified" means a protein that is in aform that is relatively free from contaminating lipids, proteins,nucleic acids or other cellular material normally associated with aprotein in a cell. A substantially purified human PTP-BAS type 4, humanPTP-BAS type 5a or mouse PTP-BAS type 5b protein can be obtained, forexample, using well known biochemical methods of purification or byexpressing a recombinant nucleic acid molecule encoding a PTP-BAS suchas the nucleic acid molecules shown as SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:22 or SEQ ID NO: 6, respectively. In addition, an amino acid sequenceconsisting of at least a portion of the amino acid sequences of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5 can be chemically synthesized or can beproduced by expressing a portion of the nucleotide sequences shown asSEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:22 or SEQ ID NO:6, respectively.

As used herein, the terms "protein" or "polypeptide" are used in thebroadest sense to mean a sequence of amino acids that can be encoded bya cellular gene or by a recombinant nucleic acid sequence or can bechemically synthesized. In some cases, the term "polypeptide" is used inreferring to a portion of an amino acid sequence encoding a full lengthprotein. An active fragment of a FAP as defined below can be an exampleof such a polypeptide. A protein can be a complete, full length geneproduct, which can be a core protein having no amino acid modificationsor can be a post-translationally modified form of a protein such as aphosphoprotein, glycoprotein, proteoglycan, lipoprotein andnucleoprotein.

In reference to a FAP such as a PTP-BAS type 4 or a PTP-BAS type 5, aprotein is characterized primarily by its ability to associate with Fas.As used herein, the term "Fas" means the full length Fas protein or aportion of the full length Fas protein such as the Fas(191-335) orFas(321-335) portions of Fas, either of which can associate with a FAP(see Example II and FIG. 6). It also should be recognized that theability of a FAP to associate with Fas can be due to a portion of thefull length FAP as is disclosed herein for PTP-BAS type 4 and for themembers of the subfamily of PTP-BAS type 5 proteins. Thus, a FAP can bean active fragment of a full length protein. As used herein, the term"active fragment" means a FAP that is a portion of a full lengthprotein, provided that the portion has an activity that ischaracteristic of the corresponding full length protein. For example, anactive fragment of a FAP such as a PTP-BAS type 4 or a PTP-BAS type 5polypeptide as disclosed herein or a GLGF2 domain of PTP-BAS can have anactivity such as the ability to bind Fas or can have an activity as animmunogen, which can be used to obtain an anti-FAP antibody. Thus, theinvention also provides active fragments of a PTP-BAS type 4 or a memberof the subfamily of PTP-BAS type 5 proteins, which can be identifiedusing the assays described below. As used herein, the term "subfamily ofPTP-BAS type 5 proteins" means PTP-BAS proteins that do not contain acatalytic phosphatase domain (see, for example, FIG. 7). As disclosedherein, human PTP-BAS type 5a and mouse PTP-BAS type 5b are examples ofmembers of the subfamily of PTP-BAS type 5 proteins. The term "PTP-BAStype 5" is used generally herein to refer to the members of thesubfamily of PTP-BAS type 5 proteins.

The present invention also provides an anti-PTP-BAS type 4 antibody andan anti-PTP-BAS type 5 antibody, including, for example, an anti-humanPTP-BAS type 5a antibody or an anti-mouse PTP-BAS type 5b antibody. Itshould be recognized that an antibody of the invention can be specificfor an epitope that is present only in a particular type of PTP-BAS orcan be specific for an epitope that is common to more than one type ofPTP-BAS. For example, an anti-PTP-BAS type 5 antibody can be specificfor a single member of the subfamily of PTP-BAS type 5 proteins such asthe human PTP-BAS type 5a protein or can be specific for more than onemember of this subfamily. As used herein, the term "antibody" is used inits broadest sense to include polyclonal and monoclonal antibodies, aswell as polypeptide fragments of antibodies that retain a specificbinding activity for a specific antigen of at least about 1×10⁵ M⁻¹. Oneskilled in the art would know that anti-PTP-BAS type 4 antibodyfragments or anti-PTP-BAS type 5 antibody fragments such as Fab,F(ab')₂, Fv and Fd fragments can retain specific binding activity for aPTP-BAS type 4 or a PTP-BAS type 5, respectively, and, thus, areincluded within the definition of an antibody. In addition, the term"antibody" as used herein includes naturally occurring antibodies aswell as non-naturally occurring antibodies and fragments of antibodiesthat retain binding activity. Such non-naturally occurring antibodiescan be constructed using solid phase peptide synthesis, can be producedrecombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al., Science 246:1275-1281 (1989),which is incorporated herein by reference.

An anti-PTP-BAS type 4 antibody or an anti-PTP-BAS type 5 antibody canbe prepared using well known methods as described, for example, byHarlow and Lane, Antibodies: A laboratory manual (Cold Spring HarborLaboratory Press, 1988), which is incorporated herein by reference. APTP-BAS type 4 or a PTP-BAS type 5 protein or a portion of a PTP-BAStype 4 or a PTP-BAS type 5 protein can be used as an immunogen. Such animmunogen can be prepared from natural sources or produced recombinantlyor, in the case of a portion of the protein, can be chemicallysynthesized. Non-immunogenic peptides of a PTP-BAS type 4 or a PTP-BAStype 5 protein can be made immunogenic by coupling the hapten to acarrier molecule such bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH) as described, for example, by Harlow and Lane, supra,1988). In addition, a PTP-BAS type 4 or a PTP-BAS type 5 fusion proteincan be expressed as described in Example II. Such fusion protein can bereadily purified and used as an immunogen. Using these methods, variousanti-FAP antibodies have been obtained (not shown).

Polyclonal antibodies can be raised, for example, in rabbits. Inaddition, monoclonal antibodies can be obtained using well known methods(see, for example, Reed et al., Anal. Biochem. 205:70-76 (1992)), whichis incorporated herein by reference; see, also, Harlow and Lane, supra,(1988)). For example, spleen cells from an immunized mouse can be fusedto an appropriate myeloma cell line such as SP/02 myeloma cells toproduce hybridoma cells. Cloned hybridoma cell lines can be screenedusing a labelled immunogen such as PTP-BAS type 4 or type 5 to identifyclones that secrete monoclonal antibodies. Hybridomas that expressantibodies having a desirable specificity and affinity can be isolatedand utilized as a continuous source of antibodies. One skilled in theart would know that a dependable source of monoclonal antibodies isdesirable, for example, for preparing diagnostic kits as describedbelow.

The invention also provides a substantially purified nucleic acidmolecule, which encodes a mammalian PTP-BAS type 4, comprisingsubstantially the nucleotide sequence encoding human PTP-BAS type 4 a sshown in FIG. 13 (SEQ ID NO:2). In addition, the invention provides asubstantially purified nucleic acid molecule, which encodes a mammalianPTP-BAS type 5, comprising, for example, substantially the nucleotidesequence encoding human PTP-BAS type 5a as shown in FIG. 15 (SEQ ID NO:4and SEQ ID NO:22) or substantially the nucleotide sequence encodingmouse PTP-BAS type 5b as shown in FIG. 17 (SEQ ID NO:6).

As used herein, the term "substantially purified" means a nucleic acidmolecule that is in a form that is relatively free from contaminatinglipids, proteins, nucleic acids or other cellular material normallyassociated with a nucleic acid molecule in a cell. A substantiallypurified nucleic acid molecule can be obtained, for example, byrecombinant DNA methods as described herein (see, also, Sambrook et al.,Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989), which is incorporated herein by reference) or can bechemically synthesized. As used herein, the term "substantially thenucleotide sequence" means the disclosed nucleotide sequence for humanPTP-BAS type 4 (SEQ ID NO: 2), human PTP-BAS type 5a (SEQ ID NO:4 andSEQ ID NO:22) or mouse PTP-BAS type 5b (SEQ ID NO:6), as well as asimilar sequence that contains, for example, different nucleotides thanshown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22 or SEQ ID NO:6, butthat, as a result of the degeneracy of the genetic code, encodessubstantially the same amino acid sequence as shown in SEQ ID NO:1, SEQID NO:3 or SEQ ID NO:5, respectively. In addition, a nucleic acidmolecule of the invention also can encode a portion of a PTP-BAS type 4or a PTP-BAS type 5 protein, including, for example, an active fragmentof a PTP-BAS type 4 or a PTP-BAS type 5 protein.

The invention also provides a nucleotide sequence that can hybridize toa portion of a nucleic acid molecule encoding a PTP-BAS type 4 or aPTP-BAS type 5 or both PTP-BAS types 4 and 5 under relatively stringenthybridization conditions. Such a nucleotide sequence should be at leastten nucleotides in length and can be prepared, for example, byrestriction endonuclease digestion of a cloned nucleic acid moleculeencoding a PTP-BAS type 4 or a PTP-BAS type 5, by PCR amplification of aportion of the nucleic acid molecule shown in FIG. 13 (SEQ ID NO:2),FIG. 15 (SEQ ID NO:4 and SEQ ID NO:22) or FIG. 17 (SEQ ID NO:6) or bychemical synthesis. Relatively stringent hybridization conditions can bedetermined empirically or can be estimated based, for example, on therelative GC:AT content of the hybridizing nucleotide sequence and thetarget sequence, the length of the hybridizing nucleotide sequence andthe number, if any, of mismatches between the hybridizing nucleotidesequence and the target sequence. If desired, a hybridizing nucleotidesequence can be detectably labelled and used as a probe or can be usedas a primer for PCR. Methods for detectably labelling a nucleotidesequence are well known in the art (see, for example, Sambrook et al.,supra, 1989; see, also, Ausubel et al., Current Protocols in MolecularBiology (Greene Publ., NY 1989), which is incorporated herein byreference).

For convenience, the term "PTP-BAS" is used generally herein to mean anyor all isoforms of PTP-BAS, including, for example, the previously knownPTP-BAS types 1, 2 and 3 and the newly disclosed PTP-BAS types 4 and 5.In addition, a PTP-BAS can be a mutant form of a PTP-BAS, which is aPTP-BAS that contains one or a few amino acid additions, deletions orsubstitutions, provided that the mutant PTP-BAS can bind Fas. Forexample, a mutant form PTP-BAS can have a single amino acidsubstitution, which can result in the loss of protein tyrosinephosphatase activity without significantly affecting the ability of themutant PTP-BAS to bind Fas. A mutant PTP-BAS can be obtained, forexample, by site directed mutagenesis of a nucleic acid moleculeencoding a PTP-BAS and screening the mutagenized nucleic acid moleculesto identify a nucleic acid molecule that expresses a mutant PTP-BAS,which can bind Fas but lacks protein tyrosine phosphatase activity.Expression in a cell of a PTP-BAS that can bind Fas but that lackscatalytic activity, including, for example, member of the subfamily ofPTP-BAS type 5 proteins, a mutant PTP-BAS or a portion of a PTP-BAS suchas the portion of PTP-BAS type 4 disclosed herein, can alter theassociation of a catalytic PTP-BAS to Fas and, therefore, can modulatethe level apoptosis in a cell. As used herein, the term "modulate" meansincrease or decrease.

The present invention also provides another example of a FAP, the 78,000dalton glucose-regulated protein (GRP78). GRP78 is a stress-relatedprotein that is located in the endoplasmic reticulum of a cell andshares sequence homology with the major heat shock protein (Ting andLee, DNA 7:275-278 (1988); Sugawara et al., Canc. Res. 53:6001-6005(1993), each of which is incorporated herein by reference). Expressionof GRP78 in a cell can decrease the susceptibility of the cell to T cellmediated cytotoxicity. As disclosed herein, GRP78 can associate with Fasand, therefore, is a FAP. Thus, GRP78 likely can modulate apoptosis in acell based on its degree of association with Fas.

The identification of a FAP that can bind Fas, which is involved inapoptosis, provides a means to identify agents that can effectivelyalter the association of a FAP with a Fas. Thus, the present inventionprovides a screening assay useful for identifying an effective agent,which can alter the association of a FAP with Fas. Since Fas is involvedin apoptosis, the identification of such effective agents can be usefulfor modulating the level of apoptosis in a cell in a subject having apathology characterized by an increased or decreased level of apoptosis.

As used herein, the term "agent" means a chemical or biological moleculesuch as a simple or complex organic molecule, a peptide, apeptido-mimetic, a protein or an oligonucleotide that has the potentialfor altering the association of a FAP and Fas or altering the activityof a FAP. In addition, the term "effective agent" is used herein to meanan agent that can, in fact, alter the association of a FAP and Fas orcan, in fact, alter the activity of a FAP.

As used herein, the term "alter the association" means that theassociation of a FAP and Fas either is increased or is decreased due tothe presence of an effective agent. As a result of an alteredassociation of a FAP and Fas in a cell, the activity of Fas or of theFAP can be increased or decreased, thereby modulating the level ofapoptosis in the cell. As used herein, the term "alter the activity"means that the agent can increase or decrease the activity of a FAP in acell, thereby modulating the level of apoptosis in the cell. Forexample, an effective agent can increase or decrease the phosphataseactivity of a PTP-BAS, without affecting the association of the PTP-BASwith Fas.

An effective agent can act by interfering with the ability of a FAP anda Fas to associate or can act by causing the dissociation of a boundFAP-Fas complex, wherein the ratio of bound FAP-Fas complex to free FAPand Fas is related to the level of apoptosis in a cell. For example,binding of a ligand to Fas can allow Fas, in turn, to bind a FAP such asa catalytic form of a PTP-BAS. The association, for example, of Fas anda catalytic PTP-BAS can result in activation or inhibition of thephosphatase activity of PTP-BAS. In the presence of an effective agent,the association of the catalytic PTP-BAS and Fas can be altered, whichcan alter the phosphatase activity of PTP-BAS in the cell. As a resultof the altered phosphatase activity, the level of apoptosis in a cellcan be increased or decreased. Thus, the identification of an effectiveagent that alters the association of Fas and a FAP can allow for the useof the effective agent to increase or decrease the level of apoptosis ina cell.

An effective agent can be useful, for example, to increase the level ofapoptosis in a cell such as a cancer cell, which is characterized byhaving a decreased level of apoptosis as compared to its normal cellcounterpart. An effective agent also can be useful, for example, todecrease the level of apoptosis in a cell such as a T lymphocyte in asubject having a viral disease such as acquired immunodeficiencysyndrome, which is characterized by an increased level of apoptosis inan infected T cell as compared to a normal T cell. Thus, an effectiveagent can be useful as a medicament for altering the level of apoptosisin a subject having a pathology characterized by increased or decreasedapoptosis. In addition, an effective agent can be used, for example, todecrease the level of apoptosis and, therefore, increase the survivaltime of a cell such as a hybridoma cell in culture. The use of aneffective agent to prolong the survival of a cell in vitro cansignificantly improve bioproduction yields in industrial tissue cultureapplications.

A PTP-BAS that lacks catalytic activity but retains the ability toassociate with Fas is an example of an effective agent, since theexpression of a non-catalytic PTP-BAS in a cell can alter theassociation of a catalytic PTP-BAS and Fas. Thus, it should berecognized that a FAP can be an effective agent, depending, for example,on the normal FAP-Fas association that occurs in a particular cell type.In addition, an active fragment of a PTP-BAS or of GRP78 can be aneffective agent, provided the active fragment can alter the associationof a FAP and Fas in a cell. Such active fragments, which can be peptidesas small as about five amino acids, can be identified, for example, byscreening a peptide library (see, for example, Ladner et al., U.S. Pat.No. 5,223,409, which is incorporated herein by reference) to identifypeptides that can bind Fas.

Similarly, a peptide or polypeptide portion of Fas also can be aneffective agent. For example, the C-terminal fifteen amino acids of Fascan associate with a FAP (see Example II; FIG. 8). A peptide such as theC-terminal peptide of Fas can be useful, for example, for decreasing theassociation of a FAP and Fas in a cell by competing for binding to theFAP. A non-naturally occurring peptido-mimetic also can be useful as aneffective agent. Such a peptido-mimetic can include, for example, apeptoid, which is peptide-like sequence containing N-substitutedglycines, or an oligocarbamate. A peptido-mimetic can be particularlyuseful as an effective agent due, for example, to having an increasedstability to enzymatic degradation in vivo.

A screening assay to identify an effective agent can be performed invivo using the two hybrid system or can be performed in vitro asdisclosed herein. The yeast two hybrid system, for example, can be usedto screen a panel of agents to identify effective agents that alter theassociation of Fas and a FAP. An effective agent can be identified bydetecting an altered level of transcription of a reporter gene. Forexample, the level of transcription of a reporter gene due to thebridging of a DNA-binding domain and trans-activation domain by Fas andFAP hybrids can be determined in the absence and in the presence of anagent. An effective agent, which alters the association between Fas anda FAP, can be identified by a proportionately altered level oftranscription of the reporter gene as compared to the control level oftranscription in the absence of the agent.

In some cases, an agent may not be able to cross the yeast cell walland, therefore, cannot enter a yeast cell to alter the association of aFAP and Fas. The use of yeast spheroplasts, which are yeast cells thatlack a cell wall, can circumvent this problem (Smith and Corcoran, InCurrent Protocols in Molecular Biology (ed. Ausubel et al.; GreenePubl., N.Y. 1989), which is incorporated herein by reference). Inaddition, an agent, upon entering a cell, may require "activation" by acellular mechanism, which may not be present in yeast. Activation of anagent can include, for example, metabolic processing of the agent or amodification such as phosphorylation of the agent, which can benecessary to convert the agent into an effective agent. In this case, amammalian cell line can be used to screen a panel of agents. Atranscription assay such as the yeast two hybrid system described inExample I can be adapted for use in mammalian cells using well knownmethods (see, for example, Fearon et al., Proc. Natl. Acad. Sci., USA89:7958-7962 (1992), which is incorporated herein by reference; see,also, Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).

The present invention also provides in vitro screening assays. Suchscreening assays are particularly useful in that they can be automated,which allows for high through-put screening, for example, of randomly orrationally designed agents such as drugs, peptido-mimetics or peptidesin order to identify those agents that effectively alter the associationof a FAP and Fas or the activity of a FAP and, thereby, modulateapoptosis. An in vitro screening assay can utilize, for example, a FAPor a FAP fusion protein such as a FAP-glutathione-S-transferase fusionprotein (GST/FAP; see Example II). For use in the in vitro screeningassay, the FAP or FAP fusion protein should have an affinity for a solidsubstrate as well as the ability to associate with Fas. For example,when a FAP is used in the assay, the solid substrate can contain acovalently attached anti-FAP antibody. Alternatively, a GST/FAP fusionprotein can be used in the assay and the solid substrate can containcovalently attached glutathione, which is bound by the GST component ofthe GST/FAP fusion protein. Similarly, Fas or a GST/Fas fusion proteincan be used in an in vitro assay as described herein.

An in vitro screening assay can be performed by allowing a FAP orFAP-fusion protein, for example, to bind to the solid support, thenadding Fas and an agent to be tested. Control reactions, which do notcontain an agent, can be performed in parallel. Following incubationunder suitable conditions, which include, for example, an appropriatebuffer concentration and pH and time and temperature that permit bindingof the particular FAP and Fas, the amount of a FAP and Fas that haveassociated in the absence of an agent and in the presence of an agentcan be determined. The association of a FAP and Fas can be detected, forexample, by attaching a detectable moiety such as a radionuclide or afluorescent label to Fas and measuring the amount of label that isassociated with the solid support, wherein the amount of label detectedindicates the amount of association of Fas and a FAP. By comparing theamount of specific binding in the presence of an agent as compared tothe control level of binding, an effective agent, which alters theassociation of a FAP and Fas, can be identified. Such an assay isparticularly useful for screening a panel of agents such as a peptidelibrary in order to detect an effective agent.

The invention further provides methods for modulating apoptosis in acell by introducing into the cell a nucleic acid molecule encoding a FAPor Fas or an antisense nucleotide sequence, which is complementary to aregion of a gene encoding a FAP or Fas and can hybridize to the gene orto an mRNA transcribed from the gene. The level of apoptosis in a cellcan be modulated by increasing or decreasing the expression of a FAP orFas in a cell, which can alter the normal steady-state association of aFAP and Fas, thereby increasing or decreasing the activity of the FAP orof Fas in the cell. Thus, a nucleic acid molecule or an antisensenucleotide sequence as described above can be useful as a medicament fortreating a pathology characterized by an increased or decreased level ofapoptosis in a cell as compared to its normal cell counterpart.

A nucleic acid sequence encoding a FAP such as a PTP-BAS or GRP78 or anactive fragment of a FAP can be expressed in a cell using well knownexpression vectors and gene transfer methods Sambrook et al., supra,(1989). Viral vectors that are compatible with a targeted cell areparticularly useful for introducing a nucleic acid encoding a FAP into acell. For example, recombinant adenoviruses having general ortissue-specific promoters can be used to deliver a nucleic acid encodinga FAP into a variety of cell types in various tissues and can directexpression of the nucleic acid in the target cell. Recombinantadeno-associated viruses also are useful for introducing a nucleic acidencoding a FAP into a cell and have the added advantage that therecombinant virus can stably integrate into the chromatin of evenquiescent non-proliferating cells such as neurons of the central andperipheral nervous systems (Lebkowski et al., Mol. Cell. Biol.8:3988-3996 (1988), which is incorporated herein by reference).

Such viral vectors are particularly useful where it is desirable tointroduce a nucleic acid encoding a FAP into a cell in a subject, forexample, for gene therapy. Viruses are specialized infectious agentsthat can elude host defense mechanisms and can infect and propagate inspecific cell types. In particular, the specificity of viral vectors forparticular cell types can be utilized to target predetermined celltypes. Thus, the selection of a viral vector will depend, in part, onthe cell type to be targeted. For example, if a neurodegenerativedisease is to be treated by increasing the level of a FAP in neuronalcells affected by the disease, then a viral vector that targets neuronalcells can be used. A vector derived from a herpes simplex virus is anexample of a viral vector that targets neuronal cells (Battleman et al.,J. Neurosci. 13:941-951 (1993), which is incorporated herein byreference). Similarly, if a disease or pathological condition of thehematopoietic system is to be treated, then a viral vector that isspecific for a particular blood cell or its precursor cell can be used.A vector based on a human immunodeficiency virus is an example of such aviral vector (Carroll et al., J. Cell. Biochem. 17E:241 (1993), which isincorporated herein by reference). In addition, a viral vector or othervector can be constructed to express a nucleic acid encoding a FAP in atissue specific manner by incorporating a tissue-specific promoter orenhancer into the vector (Dai et al., Proc. Natl. Acad. Sci. USA89:10892-10895 (1992), which is incorporated herein by reference).

Retroviral vectors can be particularly useful for introducing a nucleicacid encoding a FAP into a cell in vivo. Retroviral vectors can beconstructed either to function as infectious particles or to undergoonly a single initial round of infection. In the former case, the genomeof the virus is modified so that it maintains the necessary genes,regulatory sequences and packaging signals to synthesize new viralproteins and RNA. However, genes conferring oncogenic potential of theseviruses is destroyed. After the viral proteins are synthesized, the hostcell packages the RNA into new viral particles, which can undergofurther rounds of infection. The viral genome also is engineered toencode and express the desired recombinant gene.

In the case of non-infectious viral vectors, the helper virus genome canbe mutated to destroy the viral packaging signal required to encapsulatethe RNA into viral particles. However, the helper virus retainsstructural genes required to package a co-introduced recombinant viruscontaining a gene of interest. Without a packaging signal, a viralparticle will not contain a genome and, thus, cannot proceed throughsubsequent rounds of infection. Methods for constructing and using viralvectors are known in the art and reviewed, for example, in Miller andRosman, Biotechniques 7:980-990 (1992), which is incorporated herein byreference. The specific type of vector will depend upon the intendedapplication. These vectors are well known and readily available withinthe art or can be constructed by one skilled in the art.

For gene therapy, a vector containing a nucleic acid encoding a FAP orFas or an antisense nucleotide sequence can be administered to a subjectby various methods. For example, if viral vectors are used,administration can take advantage of the target specificity of thevectors. In such cases, there in no need to administer the vectorlocally at the diseased site. However, local administration can be aparticularly effective method of administering a nucleic acid encoding aFAP. In addition, administration can be via intravenous or subcutaneousinjection into the subject. Following injection, the viral vectors willcirculate until they recognize host cells with the appropriate targetspecificity for infection. Injection of viral vectors into the spinalfluid also can be an effective mode of administration, for example, intreating a neurodegenerative disease.

Receptor-mediated DNA delivery approaches also can be used to deliver anucleic acid molecule encoding a FAP into cells in a tissue-specificmanner using a tissue-specific ligand or an antibody that isnon-covalently complexed with the nucleic acid molecule via a bridgingmolecule (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987), each of which is incorporated hereinby reference). Direct injection of a naked or a nucleic acid moleculeencapsulated, for example, in cationic liposomes also can be used forstable gene transfer into non-dividing or dividing cells in vivo (Ulmeret al., Science 259:1745-1748 (1993), which is incorporated herein byreference). In addition, a nucleic acid molecule encoding a FAP can betransferred into a variety of tissues using the particle bombardmentmethod (Williams et al.,Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991),which is incorporated herein by reference). Such nucleic acid moleculescan be linked to the appropriate nucleotide sequences required fortranscription and translation.

A particularly useful mode of administration of a nucleic acid encodinga FAP is by direct inoculation locally at the site of the disease orpathological condition. Local administration can be advantageous becausethere is no dilution effect and, therefore, the likelihood that amajority of the targeted cells will be contacted with the nucleic acidmolecule is increased. Thus, local inoculation can alleviate thetargeting requirement necessary with other forms of administration and,if desired, a vector that infects all cell types in the inoculated areacan be used. If expression is desired in only a specific subset of cellswithin the inoculated area, then a promotor, an enhancer or otherexpression element specific for the desired subset of cells can belinked to the nucleic acid molecule. Vectors containing such nucleicacid molecules and regulatory elements can be viral vectors, viralgenomes, plasmids, phagemids and the like. Transfection vehicles such asliposomes also can be used to introduce a non-viral vector intorecipient cells. Such vehicles are well known in the art.

An alternative method of modulating apoptosis in a cell is to introducea nucleotide sequence encoding an antisense FAP or Fas into the cell andexpressing the antisense nucleotide sequence in the cell. Such anucleotide sequence can be introduced into and expressed in a cell usingthe methods and vectors described above. Chemically synthesizednucleotide sequences also can be administered directly to cells.Synthetic antisense oligonucleotides can be prepared using well knownmethods or can be purchased from commercial sources and, if desired, canincorporate nucleotide analogs to increase the resistance of theoligonucleotic to degradation by nucleases in a cell.

The present invention also provides methods for diagnosing a pathologythat is characterized by an increased or decreased level of apoptosis ina cell to determine whether the increased or decreased level ofapoptosis is due, for example, to increased or decreased expression of aFAP in the cell or to expression of a variant FAP. The identification ofsuch a pathology, which can be due to altered association of a FAP andFas in a cell, can allow for intervention therapy using an effectiveagent or a nucleic acid molecule or an antisense nucleotide sequence asdescribed above. In general, a test sample can be obtained from asubject having a pathology characterized by increased or decreasedapoptosis and can be compared to a control sample from a normal subjectto determine whether a cell in the test sample has, for example,increased or decreased expression of FAP. The level of a FAP in a cellcan be determined by contacting a sample with a reagent such as ananti-FAP antibody or Fas, either of which can specifically bind a FAP.For example, the level of a FAP in a cell can determined by well knownimmunoassay or immunohistochemical methods using an anti-FAP antibody(see, for example, Reed et al., supra, 1992; see, also, Harlow and Lane,supra, (1988)). As used herein, the term "reagent" means a chemical orbiological molecule that can specifically bind to a FAP or to Fas or toa bound FAP-Fas complex. For example, either an anti-FAP antibody or Fascan be a reagent for a FAP, whereas either an anti-Fas antibody or a FAPcan be a reagent for Fas.

As used herein, the term "test sample" means a cell or tissue specimenthat is obtained from a subject and is to be examined for expression ofa FAP in a cell in the sample. A test sample can be obtained, forexample, during surgery or by needle biopsy and can be examined usingthe methods described herein to diagnose a pathology characterized byincreased or decreased apoptosis. Increased or decreased expression of aFAP in a cell in a test sample can be determined by comparison to anexpected normal level for a FAP in a particular cell type. A normalrange of FAP levels in various cell types can be determined by samplinga statistically significant number of normal subjects. In addition, acontrol sample can be evaluated in parallel with a test sample in orderto determine whether a pathology characterized by increased or decreasedapoptosis is due to increased or decreased expression of a FAP. The testsample can be examined using, for example, immunohistochemical methodsas described above or the sample can be further processed and examined.For example, an extract of a test sample can be prepared and examined todetermine whether a FAP that is expressed in a cell in the sample canassociate with Fas in the same manner as a FAP from a control cell orwhether, instead, a variant FAP is expressed in the cell.

A diagnostic assay kit incorporating a reagent such as an anti-FAPantibody or Fas can be useful for detecting a pathology due to alteredFAP expression in a cell. Such a kit is particularly useful because itallows for standardization of assay conditions. A kit can contain, inaddition to a reagent, a reaction cocktail that provides suitablereaction conditions for performing the assay and control sample thatcontains a known amount of a FAP. In addition, the kit can contain anantibody that is specific for the reagent.

A diagnostic assay should include a simple method for detecting theamount of a FAP in a sample that is bound to the reagent. Detection canbe performed by labelling the reagent and detecting the presence of thelabel using well known methods (see, for example, Harlow and Lane,supra, 1988; chap. 9, for labelling an antibody). A reagent can belabelled with various detectable moieties including a radiolabel, anenzyme, biotin or a fluorochrome. Materials for labelling the reagentcan be included in the diagnostic kit or can be purchased separatelyfrom a commercial source. Following contact of a labelled reagent with atest sample and, if desired, a control sample, specifically boundreagent can be identified by detecting the particular moiety.

A labelled antibody that can specifically bind the reagent also can beused to identify specific binding of an unlabelled reagent. For example,if the reagent is an anti-FAP antibody, a second antibody can be used todetect specific binding of the anti-FAP antibody. A second antibodygenerally will be specific for the particular class of the firstantibody. For example, if an anti-FAP antibody is of the IgG class, asecond antibody will be an anti-IgG antibody. Such second antibodies arereadily available from commercial sources. The second antibody can belabelled using a detectable moiety as described above. When a sample islabelled using a second antibody, the sample is first contacted with afirst antibody, then the sample is contacted with the labelled secondantibody, which specifically binds to the first antibody and results ina labelled sample.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Indentification of FAP's using the Yeast Two Hybrid System

This example demonstrates the use of the yeast two hybrid system toidentify cDNA sequences encoding proteins that can associate with thecytoplasmic domain of Fas.

A. Host/Vector Systems

Two different yeast host/plasmid vector systems, designated herein asthe "EGY48 system" and the "L40 system," were used to identify cDNAsequences encoding FAP's.

1. EGY48 system

In some experiments, S. cerevisiae strain EGY48 yeast cells were used asthe host cells for the two hybrid assays ("EGY48 system"). Strain EGY48cells have a MATα trp1 ura3 his3 LEU2::pLexAop6-LEU2 genotype. Yeastwere grown in YPD medium. As indicated, strain EGY48 cells contain theLeu2 gene linked to the LexAOp and, therefore, can grow inleucine-deficient medium in the presence of a transcriptionallycompetent LexA DNA-binding domain.

The plasmids, pEG202 (FIG. 1) and pJG4-5 (FIG. 2), were used forexpressing hybrid proteins in EGY48 yeast cells (Zervous et al., Cell72:223-232 (1993); Gyuris et al., Cell 75:791-803 (1993); Golemis etal., In Current Protocols in Molecular Biology (ed. Ausubel et al.;Green Publ.; NY 1994) each of which is incorporated herein byreference). Plasmid pEG202 was derived from plasmid LexA202+PL (Ruden etal., Nature 350:250-252 (1991); Ma and Ptashne, Cell 51:113-119 (1987),each of which is incorporated herein by reference) and containsadditional unique polylinker sites for cloning. Plasmid pEG202 wascreated by cleaving LexA202+PL at the unique SalI site, which is presentin the polylinker downstream of LexA, and inserting a 22-mer thatregenerates the SalI site and also contains novel NcoI, NotI and XhoIsites.

The 22-mer was constructed by synthesizing two oligonucleotides,5'-TCGACCATGGCGGCCGCTCGAG-3' (SEQ ID NO:7) and5'-TCGACTCGAGCGGCCGCCATGG-3' (SEQ ID NO:8) and allowing thecomplementary regions of the oligonucleotides to anneal. The 22-mer wasligated into the SalI site of LexA202+PL to create pEG202. As shown inFIG. 1, pEG202 also contains the yeast 2 micron origin of replicationand a histidine selectable marker. Expression of the LexA-fusioncassette is from the strong constitutive ADH1 promotor. Insertion of acDNA encoding an open reading frame into the EcoRI, BamHI, SalI, NcoI,NotI or XhoI site of pEG202 results in the production of a LexA fusionprotein.

The plasmid pJG4-5 was derived from a pUC plasmid and contains agalactose inducible promotor (FIG. 2). Insertion of the cDNA into theEcoRI or XhoI site results in the production of a fusion protein withthe B42 trans-activation domain and containing an SV40 nuclearlocalization signal and a hemagglutinin epitope tag (Zervous et al.,supra, 1993; Gyuris et al., supra, 1993).

A β-gal reporter gene construct used in the EGY48 system was plasmidpSH18-34, which contains the lacZ gene linked to a LexA operatorsequence (FIG. 3; Hanes and Brent, Cell 57:1275-1283 (1989); Hanes andBrent, Science 251:426-430 (1991), each of which is incorporated hereinby reference). Plasmid pSH18-34, which contains 8 copies of the LexAoperator sequence, was constructed by inserting two 78 base pairoligonucleotides formed by annealing(5'-TCGACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACG-3'; SEQ ID NO:9) and(5'-TCGACGTACTGTACATATAACCACTGGTTTTATACAGCAGTACTGTACATATAACCACTGGTTTTATATACAGCAG-3'; SEQ ID NO:10) into the XhoI site of plasmidpLR1Δ1 (West et al., Mol. Cell. Biol. 4:2467-2478 (1984), which isincorporated herein by reference). Each oligonucleotide contains fourbinding sites for the LexA DNA binding protein. A stably transformedstrain EGY48 yeast cell line containing PSH18-34 was obtained based onits ability to grow in medium lacking uracil.

2. L40 system

In some experiments, S. cerevisiae strain L40 yeast cells were used asthe host cells for the two hybrid assay ("L40 system;" Vojtek et al.,supra, 1993). Strain L40 cells have a MATa, trp1, leu2, his3, ade2,LYS2:(lexAop) 4-HIS3, URA3::(lexAop)8-lacZ genotype and are stablytransformed with histidine synthetase (HIS3) and lacZ reporter genes,both of which are under the control of a lexA operator. Strain L40 cellswere grown in YPD medium.

The plasmids, pBMT-116 (FIG. 4) and pVP-16 (FIG. 5), were used in assaysusing strain L40 cells. Plasmid pBMT-116 encodes the LexA DNA-bindingdomain under control of an ADH promotor (FIG. 4). The presence ofpBMT-116 in strain L40 cells permits the cells to grow intryptophan-deficient medium. Plasmid pVP-16 encodes a trans-activatingdomain also under control of an ADH promotor (FIG. 5). The presence ofpVP-16 in strain L40 cells permits the cells to grow inleucine-deficient medium.

B. Preparation of Vectors Encoding Hybrid Proteins

As a control and to eliminate false positive clones, a cDNA sequenceencoding a Bcl-2 protein (from pSKII-bcl-2α; Tanaka et al., J. Biol,Chem. 268:10920-10926 (1993), which is incorporated herein by reference)was also modified by PCR mutagenesis (Higuchi et al., supra, 1990) usingthe primers described below and subcloned in frame into pEG202. In orderto prevent potential targeting of expressed proteins to the nucleus,sequences corresponding to the transmembrane domain of Bcl-2 weredeleted (Tsujimoto and Croce, Proc. Natl. Alcad. Sci., USA 83:5214-5216(1986), which is incorporated herein by reference) and a stop codon wasinserted at the end of the open reading frame.

1. EGY48 system

The cDNA sequence encoding the cytoplasmic domain of human Fas (aminoacids 191 to 335; see FIG. 6; Itoh et al., supra, 1991) was modified byPCR mutagenesis (Higuchi et al., In PCR Protocols (ed. Innes et al.;Academic Press; San Diego, Calif. 1990), which is incorporated herein byreference) using the primers described below and subcloned in frame intopEG202 to produce pEG/Fas(191-335). As described above, pEG202 utilizesan ADH promoter to constitutively drive expression of a fusion proteincontaining an N-terminal LexA DNA binding domain. The cDNA sequence forthe cytoplasmic domain of human Fas was subcloned into the EcoRI site ofpEG202, in-frame with the upstream LexA sequences. Forward and reverseprimers, which contained an EcoRI site (underlined) or BclI site(italics), were as follows (bold indicates DNA encoding stop codon;TCA): (i) Fas (amino acids (aa) 191 to 335)(5'-GGAATTCAAGAGAAAGGAAGTACAG-31'; SEQ ID NO:11) and(5'-TGATCACTAGACCAAGCTTTGGAT-3'; SEQ ID NO:12); (ii) Bcl-2 (aa 1 to 218)(5'-GGAATTCATGGCGCACGCTGCGAGAAC-3'; SEQ ID NO:13) and(5'-TGATCACTTCAGAGACAGCCAC-3'; SEQ ID NO:14).

The pJG4-5 plasmid utilizes a galactose-inducible promoter (GAL1-p) toinducibly drives expression of fusion proteins containing an N-terminalB42 trans-activation domain (FIG. 2). B42 fusion proteins encodingcandidate FAP's were produced by cloning a HeLa cell cDNA library intothe EcoRI or XhoI sites of pJG4-5 (pJG/HeLa).

2. L40 system

The cDNA sequences encoding the various fragments of the cytoplasmicdomain (Itoh et al., supra, 1991) were generated by PCR (Higuchi et al.,supra, 1990) using the forward (F) and reverse (R) primers containingEcoRI (underlined) and BclI (italics) sites (bold indicates STOP codon,TCA), as follows: 1) 5'-GGAATTCAAGAGAAAGGAAGTACAG-3' (F1; SEQ ID NO:11);2) 5'-GGAATTCAAAGGCTTTGCTTCGAAAG-3' (F2; SEQ ID NO:15); 3)5'-GTGATCACGCTTCTTTCTTTCCATG-3' (R1; SEQ ID NO:16); and 4)5'-GTGATCACTAGACCAAGCTTTGGAT (R2; SEQ ID NO:12). Use of the followingcombinations of primers produced the indicated Fas fragments: 1)F1+R2=Fas(191-335); 2) F1+R1=Fas(119-290); 3) F2+R1=Fas(246-335); and 4)F2+R2=Fas(246-290). Fas(321-335) was generated by restrictionendonuclease digestion using SpeI and filled-in using T4 DNA polymeraseto generate a STOP codon. The sequence encoding Fas(119-335) wassubcloned in frame into pBMT-116 to produce pBMT/Fas(191-335).

The pVP-16 plasmid utilizes an ADH promotor to drive expression offusion proteins containing the VP-16 trans-activation domain. A cDNAlibrary was prepared by random primed synthesis of 9.5-10.5 day CD1mouse embryo poly-A⁺ mRNA using phosphorylated hexamers. The ratio ofprimer to template was adjusted to encourage multiple synthesisreactions per RNA molecule, thus generating an average first strand sizeof about 500 nucleotides. Following second strand synthesis with RNAseH, DNA polymerase I and E. coli DNA ligase, the cDNA was repaired usingT4 DNA polymerase and the following linker was attached:

5'-ATCCTCTTAGACTGCGGCCGCTCA-3' (SEQ ID NO:17) 3'-GAGAATCTGACGCCGGCGAGT-5'PO₄ (SEQ ID NO:18).

The reaction products were fractionated by agarose gel electrophoresisand DNA fragments of 350 to 700 nucleotides were collected. The DNAfragments were extracted through a Millipore filter column, then phenol:chloroform (1:1) extracted and ethanol precipitated. To generate largequantities of cDNA, the size-selected DNA was amplified using the senseprimer (SEQ ID NO:9) from above in the presence of 5 mM Mg⁺². Followingamplification to near saturation, the reaction was diluted 10x andamplification was continued for one additional cycle. Incorporation ofradiolabeled nucleotide indicated an amplification efficiency of 1.9 percycle. Initial amplifiable cDNA was estimated to total more than 10⁸molecules. The amplified DNA was incubated overnight with 10 unitsNotI/μg DNA, then cloned into pVP-16 (pVP/embryo; Vojtek et al., supra,1993)).

For sequencing NotI inserts in pVP-16, the cDNA insert is flanked by aneleven nucleotide palindrome, nine of which form GC base pairs. Theseinserts can be sequenced using a sense primer(5'-GGTACCGAGCTCAATTGCGG-3'; SEQ ID NO:19), which covers part of theNotI palindrome. Alternative, sequencing can be performed at an elevatedtemperature using Taq DNA polymerase.

C. Assay Methods

Plasmid DNA was transformed into yeast cells by the LiCl method or bythe LiOAc method (Ito et al., J. Bacteriol. 153:163-168 (1983); Gietz etal., Nucl. Acids Res. 20:1425 (1992); Schiestl et al., Curr. Genet.16:339-346 (1989), each of which is incorporated herein by reference).In experiments using the EGY48 system, transformed cells were grown incomplete minimal medium lacking uracil, tryptophan or histidine asnecessary to select for the presence of pSH, pJG or pEG derivedplasmids, respectively. In addition, growth in leucine-deficient mediumindicated formation of a transcriptionally active LexA/B42 complex. Inexperiments using the L40 system, transformed cells were grown incomplete minimal medium lacking tryptophan or leucine as necessary toselect for the presence of pBMT or pVP derived plasmids, respectively.In addition, growth in histidine-deficient medium indicated formation oftranscriptionally active LexA/VP16 complex.

1. EGY48 system

Following expression of various fusion proteins, yeast cell extractswere prepared using a spheroplast method (Smith and Corcoran, In CurrentProtocols in Molecular Biology (ed. Ausubel et al., Green Publ.; NY1989), which is incorporated herein by reference) and expression ofLexA- or B42-fusion proteins was confirmed by immunoblot assays using apolyclonal anti-LexA antiserum, which can be prepared as described byBrent and Ptashne (Nature 312:612-615 (1984), which is incorporatedherein by reference) or an anti-HA1 monoclonal antibody (clone 12CA5;Boehringer Mannheim; Indianapolis, Ind.), respectively.

EGY48 yeast cells were stably transformed with PSH18-34, which expressesthe reporter lacZ gene from a lexA operator. The cells then weretransformed with pEG/Fas(191-335) and selected for growth in minimalmedium lacking histidine. The selected colonies were transformed withpJG/HeLa cDNA library and plated on medium lacking histidine, leucineand tryptophan to identify cloned cDNA sequences that encoded candidateFAP's.

Approximately 1.7×10⁷ colonies were plated to each of 20 plates. Of the17 million yeast transformants screened using the entire cytoplasmicdomain of Fas, approximately 1210 independent colonies of cell were aLeu⁺ phenotype. Four replicas were made from all 1210 Leu⁺ transformantsand plated as follows: 1) glucose, X-gal, leucine; 2) galactose, X-gal,leucine; 3) glucose; and 4) galactose plates. Eighty-five transformantsgrew on leucine-deficient medium and exhibited galactose-inducible β-galactivity.

Twenty-seven clones were selected and were "cured" of LexA/Fas-encodingplasmids by growing the cells in histidine-containing medium. The curedcells then were mated against a panel of a-type yeast (strain RFY206{Mata, ura3, trp1, leu2}) containing various plasmids that producedeither LexA/Fas, LexA/Ras, LexA/CD40, LexA/Bcl-2(short) or LexA/lamin(see, Vojtek et al., supra, 1993; Bartels et al., Biotechniques14:920-924 (1993), which is incorporated herein by reference) fusionprotein. The mated cells were selected for growth in medium lackinghistidine and tryptophan to obtain cells that contained both a pJG/HeLaclone and a LexA-fusion protein. Fifteen clones were partially sequencedand analyzed for homology by performing a database search. Four cloneswere identical to the nucleic acid sequence encoding the 78 kDa glucoseregulated protein GRP78 (not shown; Ting and Lee, supra, 1988).

2. L40 system

The pBMT/Fas(119-335) plasmid was introduced into L40 cells, whichcontain histidine synthetase (HIS3) and b-galactosidase (lacZ; β-gal)reporter genes under the control of lexA operators. The resultingtransformants were selected for ability to grow on medium lackingtryptophan, since the pBMT-116 plasmid contains a TRP1 gene thatcomplements the defect in the host strain. L40 cells containing thepBMT/Fas(191-335) then were transformed with the pVP-16/embryo cDNAlibrary. Transformants were selected by growth on plates lackingleucine, based on the presence of a LEU2 gene in the pVP-16 vector.

Colonies expressing a cDNA sequence encoding a candidate FAP wereidentified initially by their ability to grow in medium lackinghistidine due to expression of the lexAop/HIS3 reporter gene constructin these yeast cells. Approximately 300 million transformants werescreened and 395 His+ colonies were identified. These 395 clones wereexamined further using a β-gal colorimetric filter assay (Breeden &Nasmyth, Cold Spring Harb. Symp. Ouant. Biol. 50:643-650 (1985), whichis incorporated herein by reference) to identify expression of the lacZreporter gene, which is under the control of a lexA operator. Followingthis second screening procedure, 84 positive clones were obtained.

The 84 clones were cured of LexA/Fas-encoding plasmids by growing thecells in tryptophan-containing medium. The cured cells then were matedagainst a panel of a-type yeast (strain NA-8711-A {a, leu2, his3, trp1,pho3, pho5}) plasmids containing various fusion proteins as describedabove for the EGY48 system. The mated cells were selected for growth inmedium lacking tryptophan and leucine to obtain cells that containedboth a pVP\embryo clone and a LexA-fusion protein.

Of the 84 clones selected, two reacted specifically with the LexA/Fasprotein, as determined by the β-gal filter assay (Breeden and Nasmyth,supra, 1985). DNA sequence analysis of the two clones revealed insertsof approximately 350 base pairs (bp; pVP16-31) and 380 bp (pVP16-43; seeFIG. 7). Alignment of these mouse cDNA clones with each otherdemonstrated that they represented overlapping independent cDNAsequences. The cDNA clones were sequenced and a database search revealedgreater than 95% homology with a human cDNA sequence encoding theprotein tyrosine phosphatase, PTP-BAS. In particular, the cDNA sequenceswere homologous with the second GLGF repeat in PTP-BAS (FIG. 7). Asdescribed in Example II.C., below, the cDNA inserts obtained using thetwo hybrid system were used to screen a lambda phage human cDNA library.

The pVP16-31 plasmid was cointroduced into L40 cells with a pBMT-116plasmid containing nucleic acid sequences encoding various fragments ofFas (see FIG. 8) and transcriptional activation from the lacZ gene wasassayed using the β-gal filter assay (Breeden and Nasmyth, supra, 1985).As shown in FIG. 8, fragments of Fas that included the C-terminal 15amino acids of Fas were able to bind the polypeptide encoded by the cDNAinsert in pVP16-31 and, as a result, activate transcription of the lacZreporter gene.

EXAMPLE II In Vitro Assays to identify a FAP

This example describes methods for constructing fusion proteins usefulfor detecting and characterizing a FAP in vitro.

A. Production of GST/Fas Fusion Proteins

A cloning vector containing the FLAG™ immunstag peptide, an enterokinaserecognition site and heart muscle kinase (HMK) substrate domain based onpGEX-2TX (FIG. 9; Pharmacia; Piscataway, N.J.) was used for constructionof GST/Fas fusion proteins (FIG. 6). This vector contains an inducibletac promoter, the glutathione-S-transferase (GST) gene from Schistosomajaponicum, a multiple cloning site, the FLAG™ immunstag peptide forimmunological detection and purification, a thrombin and enterokinasecleavage site located between the GST gene and the multiple cloning siteand the HMK kinase domain for radiolabeling the fusion proteins (seebelow).

A series of GST/Fas fusion proteins was produced in E. coli usingpGEX-2TX (see FIGS. 6 and 8). The nucleic acid sequences encoding thevarious Fas fragments described above were subcloned into theBluescript™ plasmid (Stratagene; San Diego, Calif.) or were directlycloned into pGEX-2TX plasmid. The nucleic acid molecules encoding theGST/Fas fusion proteins were sequenced using a Seqienase™ version 2.0DNA sequencing kit (U.S. Biochemical; Cleveland, Ohio) to confirm thatthe reading frame was correct and that no erroneous nucleotides wereintroduced during PCR amplification. The GST-fusion proteins containedthe following segments of the Fas cytoplasmic domain: (A) amino acidresidues 191-335, which corresponding to the entire cytoplasmic domain(GST/Fas(191-335)); (B) residues 191-290, which represent the regionextending from the transmembrane domain to the end of the conservedregion (GST/Fas(191-290)); (C) residues 246-335, which is a region fromthe beginning of the conserved region to the C-terminus of Fas(GST/Fas(246-335)); (D) residues 246-290, which contains the region ofFas that is conserved among the members of the TNFR family of proteins(GST/Fas(246-290)); (E) residues 191-320, which contains the entirecytoplasmic domain, except the C-terminal 15 amino acids(GST/Fas(191-320)) and F) residues 321-335, which represent theC-terminal amino acids of Fas that constitute a negative regulatorydomain (GST/Fas(321-335); see FIG. 6).

The GST/Fas fusion proteins were expressed in E. coli by inducingexpression from the tac promoter with 1 mM IPTG. Approximately 1-10 μgGST/Fas fusion protein was obtained from a 1 ml bacterial culture. TheGST/Fas fusion proteins were partially purified from bacterial lysate byaffinity chromatography on glutathione-Sepharose 4B (Sigma; St. Louis,Mo.). Analysis of the GST/Fas fusion proteins by SDS-PAGE and Coomassieblue staining confirmed that the fusion proteins purified from E. colilysates had the expected molecular weight (not shown).

In some experiments, a ³² P-labelled GST/Fas fusion protein is used todetect the presence of a FAP. A ³² P-GST/Fas is generated using bovineheart muscle kinase (Sigma). Briefly, GST/Fas is immobilized by bindingto a glutathione-Sepharose column. The bound GST/Fas is washed with 1XHMK buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl₂) and thecoated beads are resuspended in 2-3 bead volumes (vol) of 1X HMK buffercontaining a 1 unit/ml catalytic subunit of cAMP-dependent proteinkinase (Sigma), 1 mCi/ml γ ³² P!-ATP (6000 Ci/mMol, 10 mCi/ml;Dupont/NEN; Boston, Mass.) and 1 mM dithiothreitol (DTT). The kinasereaction is allowed to proceed at room temperature (RT) for 60 min, thenstopped by the addition of 1 ml HMK stop buffer (10 mM sodium phosphate,pH 8.0, 10 mM sodium pyrophosphate, 10 mM EDTA, 1 mg/ml BSA). Free γ ³²P!-ATP is removed by resuspending the reaction mixture in stop buffer,pelleting the glutathione Sepharose beads by centrifugation and removingthe buffer; these steps are repeated 4x, then the Sepharose beads arewashed at least 5x with HMK buffer. After washing, the beads areresuspended in elution buffer (5 mM glutathione, 50 mM Tris-HCl, pH 8.0,120 mM NaCl) and stored at 4° C.

B. Identification of FAP's in Various Tissues

Far western blotting (ligand blotting) was used to detect the presenceof FAP's in various cell types. In one experiment, 50 μg total proteinfrom mouse S49 T cells were analyzed. Proteins were obtained by lysingthe cells in 10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% sodiumdeoxycholate, 1% Triton X-100 and protease inhibitors as described byReed et al., Canc. Res. 51:6529-6538 (1991), which is incorporatedherein by reference, then fractionated by electrophoresis on a 12%SDS-polyacrylamide gel. Following electrophoresis, the proteins weretransferred to nitrocellulose and the nitrocellulose filter wasprocessed by a series of denaturation/renaturation steps. The filterswere incubated with HBB buffer (20 mM Hepes, pH 7.5, 5 mM MgCl₂, 1 mMKCl, 5 mM DTT) containing 6 M guanidine-HCl to denature the proteins.After 10 min incubation with gentle shaking, the solution was removedand the same procedure was repeated a second time. After 10 min, thesolution was removed and the filters were then incubated sequentiallywith HBB buffer containing 3M guanidine-HCl, 1.5M guanidine-HCl, 0.75Mguanidine-HCl and 0.38M guanidine-HCl for 5 min each, followed by a 30min incubation with shaking in HBB buffer containing 5% skim milk. Thesolution was removed and replaced with HBB buffer containing 1% skimmilk and shaking was continued for 10 min. The filters were thenincubated overnight in a solution of HBB containing 1% skim milk and 1μg/ml GST/Fas(191-335), which contains the entire Fas cytoplasmicdomain, or GST (control).

Following incubation, the filters were washed 3x with PBST buffer (137mM NaCl, 2.76 mM KCl, 4.3 mM KNa₂ HPO₄, 0.2% triton X-100) for 15 min,then transferred to a solution containing 1:1000 dilution of a mouseanti-GST monoclonal antibody (GS7; Santa Cruz Biotechnology; Santa Cruz,Calif.) in phosphate buffered saline. The filters were incubated withgentle shaking for 2 hr at RT, then were washed 3x with PBST for 10 min.Following washing, the filters were soaked in 3 ml anti-mouseIgG-alkaline phosphatase (1 mg/ml; 1:7000 dilution; Promega; Madison,Wis.) for 1 hr at RT, then washed 3x with PBST buffer and colordevelopment of the complexes was performed using NBT/BCIP (nitrobluetetrazolium/5-bromo-4, chloro-3-indolyl-phosphate).

As a preliminary attempt to identify FAS/APO-1 binding proteins, GST/Fasfusion protein containing the entire cytoplasmic domain of Fas waspurified by affinity chromatography and radiolabeled in vitro using γ³²P-ATP and the catalytic subunit of cAMP-kinase. The ³² P-GST/Fas wasincubated with the protein blots. At least three FAP's having molecularweights of 32, 30 and 18 kDa were identified in the mouse S49 T cellprotein extracts (FIG. 10). No band corresponding to the 36 kDa Fasprotein was detected. Similar sized FAP's also were detected in humanbreast (MCF7), ovarian (A278), lung (H460) and colon (Caco-2) cancercell lines and in normal mouse T cells and a mouse B cell lymphoma line(RS11846) (not shown).

2. Screening a cDNA expression library

³² P-labeled GST/Fas protein can be used for direct screening of a λgt11cDNA expression library, which can be obtained from a commercial source(Clontech; Palo Alto, Calif.) or constructed using well known methods(Sambrook et al., supra, 1989). Radiolabeled GST and GST/Fas is obtainedas described above. Phage are transferred to nitrocellulose filters,which are placed in a plastic bag. Hybridization buffer (1% milk, 20 mMHepes-KOH, pH 7.7, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl₂) is added andapproximately 250,000 cpm/ml GST/Fas (or GST) are added. Filters areincubated at 4° C., overnight, in the presence of a 20-fold molar excessof unlabeled GST protein (see, for example, Kaelin et al., supra, 1992).

Positive plaques are selected and subjected to further rounds ofscreening as above. After 3-4 rounds of screening, the cDNA inserts ofplaque purified phages are amplified by PCR using primers that flank thevector cloning site or, alternatively, are liberated from the phage byrestriction digestion of purified phage DNA. These cDNA sequences aresubcloned into the EcoRI site of the pET vector, pET5c (Novagen;Madison, Wis.), which produces T7/Protein 10 fusion proteins that are inframe with the inserted cDNA. Expression of the recombinant proteins isinduced in E. coli using IPTG, fractionated by SDS-PAGE and transferredto nitrocellulose filters. The resulting blots are analyzed using thefar western method as described above using the same GST/Fas fusionprotein that originally was used to screen the library. Clones thatproduce FAP's, which bind the GST/Fas probe, are sequenced and theiramino-acid sequences are deduced. These sequences are checked againstnucleotide and protein databases to identify novel or previouslydescribed proteins.

Positive cDNA sequences obtained through the protein interaction cloningtechnique described above are used to produce hybridization probes toscreen various tissues by northern blot analysis. Appropriate clones arethen used as hybridization probes to screen commercially-availablelambda phage cDNA libraries prepared from an appropriate tissue toobtain a cDNA encoding the entire open reading frames for various FAP's.Alternatively, 5'- or 3'-RACE methods can be used to directly amplifythe regions of the cDNA from reverse transcribed RNA (Frohman et al.,Proc. Natl. Acad. Sci., USA 85:8998-9002 (1988), which is incorporatedherein by reference).

C. Characterization of FAP binding to Fas

The ability of a FAP identified using the yeast two hybrid system asdescribed above to associate with the GST/Fas fusion proteins wasexamined. The cDNA sequence in HFAP10 was subcloned into the Bluescriptvector, pSK-II (Stratagene), and was translated in vitro from aninternal methionine codon in the presence of ³⁵ S-L-methionine using acoupled in vitro transcription/translation system (TNT lysate; Promega)and T7 RNA polymerase. The ³⁵ S-labeled protein was incubated withvarious GST/Fas fusion proteins, which had been immobilized onglutathione-Sepharose beads in buffer A (150 mM NaCl, 50 mM Tris, pH8.0, 5 mM DTT, 2 mM EDTA, 0.1% NP-40, 1 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin) for 16 hrs at 4° C. Following incubation,the beads were washed vigorously 10x in buffer A and bound proteins wererecovered with the glutathione-Sepharose beads by centrifugation, elutedinto boiling Laemmli buffer and analyzed by SDS-PAGE and fluorography.

The polypeptide derived from the human PTP-BAS bound specifically toGST/Fas(191-335), which contains the entire Fas cytoplasmic domain, toGST/Fas(246-335), which contains the conserved region of Fas to theC-terminus and to GST/Fas(321-335), which contains the C-terminal 15amino acids of Fas (FIG. 8; "In vitro binding assay"). In contrast, thepolypeptide did not bind to GST/Fas fusion proteins containing theconserved region, alone, to the region of Fas beginning from thetransmembrane domain to the end of the conserved region, to GST, alone,to either of two forms of the TNFR or to CD40 (not shown). These resultscorrelate with the results of the two hybrid assay described above andindicate that the C-terminal 15 amino acids of Fas are sufficient forthe association of Fas with a FAP such as PTP-BAS and that FAP'sassociate specifically with Fas, but not other members of the TNFRfamily.

EXAMPLE III Characterization of PTP-BAS Type 4 and PTP-BAS Type 5a

This example describes the characteristics of human PTP-BAS type 4 andhuman PTP-BAS type 5a.

Clones pVP16-31 and pVP16-41 (see above) were used as hybridizationprobes to screen a human fetal brain cDNA library prepared in lambdagt11 (Clontech) and a mouse liver cDNA library (see Example IV). Fromthe human cDNA library, two clones were obtained (FIG. 7; HFAP10 andHFAP20). The cDNA inserts were sequenced as described above and theamino acid sequences were deduced (see FIGS. 12 and 14). Thepolypeptides encoded by these cDNA sequences were designated PTP-BAStype 4 and PTP-BAS type 5a, respectively.

HFAP10 contained an 1814 nucleotide insert (FIG. 13; SEQ ID NO:2), whichencoded 604 amino acids of PTP-BAS type 4 (FIG. 12; SEQ ID NO:1). Asshown in FIG. 11, the amino acid sequence of PTP-BAS type 4 comprisesamino acids 1279-1883 relative to the amino acid residues of PTP-BAStype 1. This amino acid sequence of PTP-BAS type 4 includes GLGF2, GLGF3and GLGF4. In addition, PTP-BAS type 4 includes a 5 amino acid VLFDKinsert in GLGF2.

HFAP20 contained an approximately 1500 nucleotide insert, of which about600 nucleotides have yet to be determined (FIG. 15; SEQ ID NO:4and SEQID NO:22). The nucleotide sequence of HFAP20 encoded a 92 amino acidpolypeptide. The N-terminal 68 amino acids of the polypeptide encoded byHFPA20 are homologous to PTP-BAS and, therefore, the polypeptide hasbeen designated PTP-BAS type 5a (FIG. 14; SEQ ID NO:3). As shown in FIG.11, the amino acid sequence of PTP-BAS type 5a comprises amino acids1377-1445 relative to the amino acid residues of PTP-BAS type 1 andincludes a GLGF2, which, like PTP-BAS type 4, contains a 5 amino acidVLFDK (SEQ ID NO:21) insert in GLGF2. The remaining 24 C-terminal aminoacids in the PTP-BAS -type 5a polypeptide are not homologous to theother members of the PTP-BAS family of proteins, including PTP-BAS type4 (see FIG. 11). Significantly, the amino acid sequence is terminated bya STOP codon, TGA, (nucleotides 4408-4410 in FIG. 15). As a result,PTP-BAS type 5a does not contain a catalytic phosphatase domain (seeFIG. 11). As described below, human PTP-BAS type 5a is a member of asubfamily of PTP-BAS type 5 proteins, which do not contain a catalyticphosphatase domain.

EXAMPLE IV Identification and Characterization of Mouse PTP-BAS Type 5b

This example describes the identification and characterization of mousePTP-BAS type 5b, which is a member of the subfamily of PTP-BAS typeproteins using the yeast two hybrid system.

The cDNA inserts from plasmids pVP16-31 and pVP16-43 were used to screena mouse liver cDNA library, which was cloned in lambda gt11 (Clontech).A cDNA clone was obtained that contained an insert encoding a mouse FAP,MFAP23 (FIG. 11). A partial nucleotide sequence for MFAP23 is shown inFIG. 17 (SEQ ID NO:6) and the deduced amino acid sequence is shown inFIG. 16 (SEQ ID NO:5).

As shown in FIG. 11, the N-terminus of the amino acid sequence encodedby MFAP23 is homologous with the amino acid sequence of a PTP-BASbeginning with amino acid residue 1180 in the human PTP-BAS type 1protein. The mouse PTP-BAS contains GFLG2, GLGF3, GLGF4 and GLGF5repeats. However, like human PTP-BAS type 5a, the mouse PTP-BAS type 5bprotein diverges at its C-terminus and terminates due to a STOP codon(see FIGS. 11, 16 and 17). As a result, the mouse PTP-BAS type 5b doesnot contain a catalytic phosphatase domain. Thus, the mouse PTP-BAS isanother example of a member of a subfamily of PTP-BAS type 5 proteins,which do not contain a catalytic phosphatase domain.

Although the invention has been described with reference to the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 22                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 610 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       HisGlySerProSerProSerValIleSerLysAlaThrGluLysGlu                              151015                                                                        ThrPheThrAspSerAsnGlnSerLysThrLysLysProGlyIleSer                              202530                                                                        AspValThrAspTyrSerAspArgGlyAspSerAspMetAspGluAla                              354045                                                                        ThrTyrSerSerSerGlnAspHisGlnThrProLysGlnGluSerSer                              505560                                                                        SerSerValAsnThrSerAsnLysMetAsnPheLysThrPheSerSer                              65707580                                                                      SerProProLysProGlyAspIlePheGluValGluLeuAlaLysAsn                              859095                                                                        AspAsnSerLeuGlyIleSerValThrValLeuPheAspLysGlyGly                              100105110                                                                     ValAsnThrSerValArgHisGlyGlyIleTyrValLysAlaValIle                              115120125                                                                     ProGlnGlyAlaAlaGluSerAspGlyArgIleHisLysGlyAspArg                              130135140                                                                     ValLeuAlaValAsnGlyValSerLeuGluGlyAlaThrHisLysGln                              145150155160                                                                  AlaValGluThrLeuArgAsnThrGlyGlnValValHisLeuLeuLeu                              165170175                                                                     GluLysGlyGlnSerProThrSerLysGluHisValProValThrPro                              180185190                                                                     GlnCysThrLeuSerAspGlnAsnAlaGlnGlyGlnGlyProGluLys                              195200205                                                                     ValLysLysThrThrGlnValLysAspTyrSerPheValThrGluGlu                              210215220                                                                     AsnThrPheGluValLysLeuPheLysAsnSerSerGlyLeuGlyPhe                              225230235240                                                                  SerPheSerArgGluAspAsnLeuIleProGluGlnIleAsnAlaSer                              245250255                                                                     IleValArgValLysLysLeuPheProGlyGlnProAlaAlaGluSer                              260265270                                                                     GlyLysIleAspValGlyAspValIleLeuLysValAsnGlyAlaSer                              275280285                                                                     LeuLysGlyLeuSerGlnGlnGluValIleSerAlaLeuArgGlyThr                              290295300                                                                     AlaProGluValPheLeuLeuLeuCysArgProProProGlyValLeu                              305310315320                                                                  ProGluIleAspThrAlaLeuLeuThrProLeuGlnSerProAlaGln                              325330335                                                                     ValLeuProAsnSerSerLysAspSerSerGlnProSerCysValGlu                              340345350                                                                     GlnSerThrSerSerAspGluAsnGluMetSerAspLysSerLysLys                              355360365                                                                     GlnCysLysSerProSerArgArgAspSerTyrSerAspSerSerGly                              370375380                                                                     SerGlyGluAspAspLeuValThrAlaProAlaAsnIleSerAsnSer                              385390395400                                                                  ThrTrpSerSerAlaLeuHisGlnThrLeuSerAsnMetValSerGln                              405410415                                                                     AlaGlnSerHisHisGluAlaProLysSerGlnGluAspThrIleCys                              420425430                                                                     ThrMetPheTyrTyrProGlnLysIleProAsnLysProGluPheGlu                              435440445                                                                     AspSerAsnProSerProLeuProProAspMetAlaProGlyGlnSer                              450455460                                                                     TyrGlnProGlnSerGluSerAlaSerSerSerSerMetAspLysTyr                              465470475480                                                                  HisIleHisHisIleSerGluProThrArgGlnGluAsnTrpThrPro                              485490495                                                                     LeuLysAsnAspLeuGluAsnHisLeuGluAspPheGluLeuGluVal                              500505510                                                                     GluLeuLeuIleThrLeuIleLysSerGluLysGlySerLeuGlyPhe                              515520525                                                                     ThrValThrLysGlyAsnGlnArgIleGlyCysTyrValHisAspVal                              530535540                                                                     IleGlnAspProAlaLysSerAspGlyArgLeuLysProGlyAspArg                              545550555560                                                                  LeuIleLysValAsnAspThrAspValThrAsnMetThrHisThrAsp                              565570575                                                                     AlaValAsnLeuLeuArgAlaAlaSerLysThrValArgLeuValIle                              580585590                                                                     GlyArgValLeuGluLeuProArgIleProMetLeuProHisLeuLeu                              595600605                                                                     ProAsp                                                                        610                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1830 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CATGGCAGCCCTTCCCCATCTGTAATATCCAAAGCCACCGAGAAAGAGACTTTCACTGAT60                AGTAACCAAAGCAAAACTAAAAAGCCAGGCATTTCTGATGTAACTGATTACTCAGACCGT120               GGAGATTCAGACATGGATGAAGCCACTTACTCCAGCAGTCAGGATCATCAAACACCAAAA180               CAGGAATCTTCCTCTTCAGTGAATACATCCAACAAGATGAATTTTAAAACTTTTTCTTCA240               TCACCTCCTAAGCCTGGAGATATCTTTGAGGTTGAACTGGCTAAAAATGATAACAGCTTG300               GGGATAAGTGTCACGGTACTGTTTGACAAGGGAGGTGTGAATACGAGTGTCAGACATGGT360               GGCATTTATGTGAAAGCTGTTATTCCCCAGGGAGCAGCAGAGTCTGATGGTAGAATTCAC420               AAAGGTGATCGCGTCCTAGCTGTCAATGGAGTTAGTCTAGAAGGAGCCACCCATAAGCAA480               GCTGTGGAAACACTGAGAAATACAGGACAGGTGGTTCATCTGTTATTAGAAAAGGGACAA540               TCTCCAACATCTAAAGAACATGTCCCGGTAACCCCACAGTGTACCCTTTCAGATCAGAAT600               GCCCAAGGTCAAGGCCCAGAAAAAGTGAAGAAAACAACTCAGGTCAAAGACTACAGCTTT660               GTCACTGAAGAAAATACATTTGAGGTAAAATTATTTAAAAATAGCTCAGGTCTAGGATTC720               AGTTTTTCTCGAGAAGATAATCTTATACCGGAGCAAATTAATGCCAGCATAGTAAGGGTT780               AAAAAGCTCTTTCCTGGACAGCCAGCAGCAGAAAGTGGAAAAATTGATGTAGGAGATGTT840               ATCTTGAAAGTGAATGGAGCCTCTTTGAAAGGACTATCTCAGCAGGAAGTCATATCTGCT900               CTCAGGGGAACTGCTCCAGAAGTATTCTTGCTTCTCTGCAGACCTCCACCTGGTGTGCTA960               CCGGAAATTGATACTGCGCTTTTGACCCCACTTCAGTCTCCAGCACAAGTACTTCCAAAC1020              AGCAGTAAAGACTCTTCTCAGCCATCATGTGTGGAGCAAAGCACCAGCTCAGATGAAAAT1080              GAAATGTCAGACAAAAGCAAAAAACAGTGCAAGTCCCCATCCAGAAGAGACAGTTACAGT1140              GACAGCAGTGGGAGTGGAGAAGATGACTTAGTGACAGCTCCAGCAAACATATCAAATTCG1200              ACCTGGAGTTCAGCTTTGCATCAGACTCTAAGCAACATGGTATCACAGGCACAGAGTCAT1260              CATGAAGCACCCAAGAGTCAAGAAGATACCATTTGTACCATGTTTTACTATCCTCAGAAA1320              ATTCCCAATAAACCAGAGTTTGAGGACAGTAATCCTTCCCCTCTACCACCGGATATGGCT1380              CCTGGGCAGAGTTATCAACCCCAATCAGAATCTGCTTCCTCTAGTTCGATGGATAAGTAT1440              CATATACATCACATTTCTGAACCAACTAGACAAGAAAACTGGACACCTTTGAAAAATGAC1500              TTGGAAAATCACCTTGAAGACTTTGAACTGGAAGTAGAACTCCTCATTACCCTAATTAAA1560              TCAGAAAAAGGAAGCCTGGGTTTTACAGTAACCAAAGGCAATCAGAGAATTGGTTGTTAT1620              GTTCATGATGTCATACAGGATCCAGCCAAAAGTGATGGAAGGCTAAAACCTGGGGACCGG1680              CTCATAAAGGTTAATGATACAGATGTTACTAATATGACTCATACAGATGCAGTTAATCTG1740              CTCCGGGCTGCATCCAAAACAGTCAGATTAGTTATTGGACGAGTTCTAGAATTACCCAGA1800              ATACCCATGTTGCCTCATTTGCTACCGGAC1830                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerLeuGlyIleSerValThrValLeuPheAspLysGlyGlyValAsn                              151015                                                                        ThrSerValArgHisGlyGlyIleTyrValLysAlaValIleProGln                              202530                                                                        GlyAlaAlaGluSerAspGlyArgIleHisLysGlyAspArgValLeu                              354045                                                                        AlaValAsnGlyValSerLeuGluGlyAlaThrHisLysGlnAlaVal                              505560                                                                        GluThrLeuArgAsnThrGlyGlnValThrAspHisTyrThrAsnLeu                              65707580                                                                      LeuGlnTyrLeuArgArgAlaLysGlnCysValAsnAsnIleSerSer                              859095                                                                        His                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 712 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGCTTGGGGATAAGTGTCACGGTACTGTTTGACAAGGGAGGTGTGAATACGAGTGTCAGA60                CATGGTGGCATTTATGTGAAAGCTGTTATTCCCCAGGGAGCAGCAGAGTCTGATGGTAGA120               ATTCACAAAGGTGATCGCGTCCTAGCTGTCAATGGAGTTAGTCTAGAAGGAGCCACCCAT180               AAGCAAGCTGTGGAAACACTGAGAAATACAGGACAGGTAACAGATCATTATACCAACCTT240               TTACAGTACCTTAGAAGAGCAAAACAATGTGTGAATAACATCAGTTCTCATTGAGATCTC300               TAAATTTGTCAGCTAATCAAGAAACCAAGCCTGATATATATAACCATCTGGGTTGTTGAT360               TTTTCCTTCCAAATTGAAATGCAAGTATTACAAGACATTTTTTACTGAGGAAGCTGACTT420               TCTATGTCACATTTAACGTTACATTACCAAAGAGATCTGATGGGGGAGGGATGGAAATTG480               CATTTTAAATTTGTTGTATAAACATCTCATTTCTAGTGGTTTTCACTCTTATTCTTTAGC540               CTTAACACAAAATTTATTTTGTTGAAGTACATTTTGAGTTAGGGAGTTTAACCAAATTAT600               CTATAATGGTCTTTGGAGGAAAAAGTTGTTGTTTTGAGACAGGGTGTTGCTGTGAGGCCC660               AGGCTGGAGTGCAGTGGCGCAATCACGGCTCACTGCAACCTTGACTTCCCAG712                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgAlaAlaIleSerAlaProArgPheThrLysAlaAsnGlyLeuThr                              151015                                                                        SerMetGluProSerGlyGlnProAlaLeuMetProLysAsnSerPhe                              202530                                                                        SerLysAlaArgThrLysProPhePheGlnValIleAlaIlePheAsn                              354045                                                                        AsnGlnCysAlaTyrValSerTyrGlnIleAspPheIleIleLysCys                              505560                                                                        SerSerAspThrCys                                                               65                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 258 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGAGCTGCCATTTCTGCGCCCAGGTTCACCAAAGCCAACGGCCTAACCAGCATGGAGCCT60                TCTGGACAGCCTGCACTCATGCCCAAGAACTCCTTCTCCAAGGCAAGAACAAAACCTTTC120               TTTCAAGTCATAGCCATTTTTAATAACCAATGTGCTTATGTGTCATACCAAATAGATTTC180               ATAATTAAATGCTCTTCAGACACATGCTAACAGTAGGACTGCTCTGTGATGAACTAACAG240               GTTTTGCTCACACTGCAG258                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCGACCATGGCGGCCGCTCGAG22                                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TCGACTCGAGCGGCCGCCATGG22                                                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCGACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACTGCTGTATATAAAACCAGT60                GGTTATATGTACAGTACG78                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCGACGTACTGTACATATAACCACTGGTTTTATATACAGCAGTACTGTACATATAACCAC60                TGGTTTTATATACAGCAG78                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGAATTCAAGAGAAAGGAAGTACAG25                                                   (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TGATCACTAGACCAAGCTTTGGAT24                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGAATTCATGGCGCACGCTGGGAGAAC27                                                 (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TGATCACTTCAGAGACAGCCAC22                                                      (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGAATTCAAAGGCTTTGCTTCGAAAG26                                                  (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GTGATCACGCTTCTTTCTTTCCATG25                                                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATCCTCTTAGACTGCGGCCGCTCA24                                                    (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGAGCGGCCGCAGTCTAAGAG21                                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGTACCGAGCTCAATTGCGG20                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlyLeuGlyPhe                                                                  (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ValLeuPheAspLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 288 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CCTGGCTAATTTTTATATTTTTAGTAGAGATGGGGCCTCACCATGTTGGCCAGGCTGGTC60                TCCAACTTCTGACCTCAGGTGATCTGCCCACCTTGGCCTCCCAAAGTGTTAGCCTTACCA120               GCATGAGCCACTCCACCTGGCCATTATCATACATTTCTAACATGTATTATATTTATAATA180               GATTCTTTTTAATCATTTATCTTTCTATACAGAAATGTAATAAAAACTTGATTTTGGAAC240               TTTCAACCCCTTGCTTTTGTTCCTCTATTTTTTTTTTCCCGGAATTCC288                           __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule comprising the nucleotidesequence shown in FIG. 13 (SEQ ID NO:2) encoding a mammalian PTP-BAStype 4, which is related to a protein tyrosine phosphatase originallyisolated from basophils (PTP-BAS).
 2. An isolated nucleic acid moleculecomprising the nucleotide sequence shown in FIG. 15 (SEQ ID NO:4)encoding human PTP-BAS type 5a.
 3. An isolated nucleic acid moleculecomprising the nucleotide sequence shown in FIG. 17 (SEQ ID NO:6)encoding mouse PTP-BAS type 5b.
 4. An isolated nucleic acid moleculeencoding mammalian PTP-BAS type 4 comprising the amino acid sequenceshown in FIG. 12 (SEQ ID NO:1).
 5. An isolated nucleic acid moleculeencoding human PTP-BAS type 5a comprising the amino acid sequence shownin FIG. 14 (SEQ ID NO:3).
 6. An isolated nucleic acid molecule encodingmouse PTP-BAS type 5b comprising the amino acid sequence shown in FIG.16 (SEQ ID NO:5).
 7. A vector, comprising a nucleic acid moleculeselected from the group consisting of the nucleic acid molecule of claim1, claim 2, claim 3, claim 4, claim 5 and claim
 6. 8. A host cellcontaining the vector of claim
 7. 9. An isolated nucleotide sequence,comprising at least ten nucleotides that hybridizes under relativelystringent conditions to a nucleic acid molecule selected from the groupconsisting of the nucleic acid molecule of claim 1, claim 2, claim 3,claim 4, claim 5 and claim 6, wherein said relatively stringentconditions allow hybridization to a nucleic acid molecule encoding aPTP-BAS type 4 or a PTP-BAS type 5, but not to another nucleic acidmolecule.